Optimized Liver-Specific Expression Systems for FVIII and FIX

ABSTRACT

The present invention relates to nucleic acid expression cassettes and vectors containing liver-specific regulatory elements and codon-optimized factor IX or factor VIII transgenes, methods employing these expression cassettes and vectors and uses thereof. The present invention is particularly useful for applications using liver-directed gene therapy, in particular for the treatment of hemophilia A and B.

FIELD OF THE INVENTION

The invention relates to nucleic acid expression cassettes andexpression vectors for gene therapy with improved liver-specificexpression capabilities, particularly for use as a gene therapy meansfor the treatment of hemophilia, more particularly for restoringcoagulation factor IX (FIX) and/or coagulation factor VIII (FVIII)deficiency in liver-directed gene therapy of respectively, hemophilia Band hemophilia A.

BACKGROUND OF THE INVENTION

Hemophilia B is an X-linked, recessive bleeding disorder caused bydeficiency of clotting factor IX (FIX). Hemophilia A is a seriousbleeding disorder caused by a deficiency in, or complete absence of, theblood coagulation factor VIII (FVIII). The clinical presentation forhemophilia A and B is characterized by episodes of spontaneous andprolonged bleeding. There are an estimated 1 in 5,000 and 1 in 20,000individuals suffer from hemophilia A and B, respectively.

Currently, hemophilia A and B is treated with protein replacementtherapy using either plasma-derived or recombinant FVIII or FIX.Although protein replacement markedly improved the life expectancy ofpatients suffering from hemophilia, they are still at risk for severebleeding episodes and chronic joint damage, since prophylactic treatmentis restricted by the short half-life, the limited availability and thehigh cost of purified clotting factors, which can approach100.000$/patient/year. In addition, the use of plasma-derived factorsobtained from contaminated blood sources increases the risk of viraltransmission. Gene therapy offers the promise of a new method oftreating hemophilia B, since the therapeutic window is relatively broadand levels slightly above 1% of normal physiologic levels aretherapeutic. If successful, gene therapy could provide constant FVIII orFIX synthesis which may lead to a cure for this disease. The differentmodalities for gene therapy of hemophilia have been extensively reviewed(Chuah et al., 2012a, 2012b, 2012c; VandenDriessche et al., 2012; High2001, 2011; Matrai et al., 2010a, 2010b).

The severity of hemophilia A and hemophilia B has been classified by thesubcommittee on Factor VIII and Factor IX of the Scientific andStandardization Committee of the International Society on Thrombosis andHaemostasis into three forms, depending on respectively, the FVIII leveland the FIX level: 1) severe form (FVIII or FIX level less than 0.01international units (IU)/ml, i.e. less than 1% of normal FVIII or FIXlevel), 2) moderate form (FVIII or FIX level from 0.01 to 0.05 IU/ml,i.e. from 1 to 5% of normal FVIII or FIX level), and 3) mild from (FVIIIor FIX level higher than 0.05 to 0.4 IU/ml, i.e. higher than 5 to 40% ofnormal FVIII or FIX level). Hemophilia A is the most common hereditarycoagulation disorder with an incidence approaching approximately 1 in5000 males.

Protein substitution therapy (PST) with purified or recombinant FVIIIand FIX has significantly improved the patients' quality of life.However, PST is not curative and patients are still at risk ofdeveloping potentially life-threatening hemorrhages and crippling jointinflammation. Unfortunately, many patients suffering from hemophilia A(up to 40%) develop neutralizing antibodies to FVIII (i.e. “inhibitors”)following PST. Similarly, an estimated 10% of patients suffering fromhemophilia B develop “inhibitors” to FIX. These inhibitors complicatethe management of bleeding episodes and can render further PSTineffective. These limitations of PST, justify the development of genetherapy as a potential alternative for hemophilia treatment.Furthermore, only a modest increase in FIX or FVIII plasma concentrationis needed for therapeutic benefit, with levels of more than 1% of normallevels able to achieve markedly reduced rates of spontaneous bleedingand long-term arthropathy.

The liver is the main physiological site of FIX and FVIII synthesis andhence, hepatocytes are well suited target cells for hemophilia genetherapy. From this location, FIX or FVIII protein can easily enter intothe circulation. Moreover, the hepatic niche may favor the induction ofimmune tolerance towards the transgene product (Annoni et al., 2007;Follenzi et al., 2004; Brown et al., 2007; Herzog et al., 1999; Matraiet al., 2011; Matsui et al., 2009). Liver-directed gene therapy forhemophilia can be accomplished with different viral vectors includingretroviral (Axelrod et al., 1990; Kay et al., 1992; VandenDriessche etal., 1999, Xu et al., 2003, 2005), lentiviral (Ward et al., 2011, Brownet al., 2007, Matrai et al., 2011), adeno-associated viral (AAV) (Herzoget al., 1999) and adenoviral vectors (Brown et al., 2004; Ehrhardt &Kay, 2002). In particular, AAV is a naturally occurring replicationdefective non-pathogenic virus with a single stranded DNA genome. AAVvectors have a favorable safety profile and are capable of achievingpersistent transgene expression. Long-term expression is predominantlymediated by episomally retained AAV genomes. More than 90% of the stablytransduced vector genomes are extra-chromosomal, mostly organized ashigh-molecular-weight concatamers. Therefore, the risk of insertionaloncogenesis is minimal, especially in the context of hemophilia genetherapy where no selective expansion of transduced cells is expected tooccur. Nevertheless, oncogenic events have been reported followingAAV-based gene transfer (Donsante et al., 2007) but it has beendifficult to reproduce these findings in other model systems (Li et al.,2011). The major limitation of AAV vectors is the limited packagingcapacity of the vector particles (i.e. approximately 5.0 kb, includingthe AAV inverted terminal repeats), constraining the size of thetransgene expression cassette to obtain functional vectors (Jiang etal., 2006). Several immunologically distinct AAV serotypes have beenisolated from human and non-human primates (Gao et al., 2002, Gao et al.2004), although most vectors for hemophilia gene therapy were initiallyderived from the most prevalent AAV serotype 2. The first clinicalsuccess of AAV-based gene therapy for congenital blindness underscoresthe potential of this gene transfer technology (Bainbridge et al.,2008). AAV-mediated hepatic gene transfer is an attractive alternativefor gene therapy of hemophilia for both liver and muscle-directed genetherapy (Herzog et al., 1997, 1999, 2002; Arruda et al., 2010; Fields etal., 2001; Buchlis et al., 2012; Jiang et al., 2006; Kay et al., 2000).Preclinical studies with the AAV vectors in murine and canine models ofhemophilia or non-human primates have demonstrated persistenttherapeutic expression, leading to partial or complete correction of thebleeding phenotype in the hemophilic models (Snyder et al., 1997, 1999;Wang et al., 1999, 2000; Mount et al., 2002; Nathwani et al., 2002).Particularly, hepatic transduction conveniently induces immune toleranceto FIX that required induction of regulatory T cells (Tregs) (Mingozziet al., 2003; Dobrzynski et al., 2006). Long-term correction of thehemophilia phenotype without inhibitor development was achieved ininhibitor-prone null mutation hemophilia B dogs treated withliver-directed AAV2-FIX gene therapy (Mount et al, 2002). In order tofurther reduce the vector dose, more potent FIX expression cassetteshave been developed. This could be accomplished by using strongerpromoter/enhancer elements, codon-optimized FIX or self-complementary,double-stranded AAV vectors (scAAV) that overcome one of the limitingsteps in AAV transduction (i.e. single-stranded to double-stranded AAVconversion) (McCarty, 2001, 2003; Nathwani et al, 2002, 2006, 2011; Wuet al., 2008). Alternative AAV serotypes could be used (e.g. AAV8 orAAV5) that result in increased transduction into hepatocytes, improveintra-nuclear vector import and may reduce the risk of T cell activation(Gao et al., 2002; Vandenberghe et al., 2006) though it is not certainthat this would necessarily also translate to human subjects since theepitopes are conserved between distinct AAV serotypes (Mingozzi et al.,2007). Liver-directed gene therapy for hemophilia B with AAV8 or AAV9 ismore efficient than when lentiviral vectors are used, at least in mice,and resulted in less inflammation (VandenDriessche et al., 2007, 2002).Furthermore, recent studies indicate that mutations of thesurface-exposed tyrosine residues allow the vector particles to evadephosphorylation and subsequent ubiquitination and, thus, preventproteasome-mediated degradation, which resulted in a 10-fold increase inhepatic expression of FIX in mice (Zhong et al., 2008).

These liver-directed preclinical studies paved the way toward the use ofAAV vectors for clinical gene therapy in patients suffering from severehemophilia B. Hepatic delivery of AAV-FIX vectors resulted in transienttherapeutic FIX levels (maximum 12% of normal levels) in subjectsreceiving AAV-FIX by hepatic artery catheterization (Kay et al., 2000).However, the transduced hepatocytes were able to present AAVcapsid-derived antigens in association with MHC class I to T cells(Manno et al., 2006, Mingozzi et al., 2007). Although antigenpresentation was modest, it was sufficient to flag the transducedhepatocytes for T cell-mediated destruction. Recently, gene therapy forhemophilia made an important step forward (Nathwani et al., 2011;Commentary by VandenDriessche & Chuah, 2012). Subjects suffering fromsevere hemophilia B (<1% FIX) were injected intravenously withself-complementary (sc) AAV8 vectors expressing codon-optimized FIX froma liver-specific promoter. This AAV8 serotype exhibits reducedcross-reactivity with pre-existing anti-AAV2 antibodies. Interestingly,its uptake by dendritic cells may be reduced compared to conventionalAAV2 vectors, resulting in reduced T cell activation (Vandenberghe etal., 2006). In mice, AAV8 allows for a substantial increase in hepatictransduction compared to AAV2, though this advantage may be lost inhigher species, like dog, rhesus monkeys and man. Subjects receivedescalating doses of the scAAV8-FIX vector, with two participants perdose. All of the treated subjects expressed FIX above the therapeutic 1%threshold for several months after vector administration, yieldingsustained variable expression levels (i.e. 2 to 11% of normal levels).The main difference with the previous liver-directed AAV trial is thatfor the first time sustained therapeutic FIX levels could be achievedafter gene therapy. Despite this progress, T-cell mediated clearance ofAAV-transduced hepatocytes remains a concern consistent with elevatedliver enzyme levels in some of the patients. Transient immunesuppression using a short course of glucocorticoids was used in anattempt to limit this vector-specific immune response.

One of the significant limitations in the generation of efficient viralgene delivery systems for the treatment of hemophilia A by gene therapyis the large size of the FVIII cDNA. Previous viral vector-based genetherapy studies for hemophilia A typically relied on the use of smallbut weak promoters, required excessively high vector doses that were notclinically relevant or resulted in severely compromised vector titers.Several other ad hoc strategies were explored, such as the use of splitor dual vector design to overcome the packaging constraints of AAV, butthese approaches were overall relatively inefficient and raisedadditional immunogenicity concerns (reviewed in Petrus et al., 2010). Ithas been found that the FVIII B domain is dispensable for procoagulantactivity. Consequently, FVIII constructs in which the B domain isdeleted are used for gene transfer purposes since their smaller size ismore easily incorporated into vectors. Furthermore, it has been shownthat deletion of the B domain leads to a 17-fold increase in mRNA andprimary translation product. FVIII wherein the B domain is deleted andreplaced by a short 14-amino acid linker is currently produced as arecombinant product and marketed as Refacto® for clinical use (WyethPharma) (Sandberg et al., 2001). Miao et al. (2004) added back a short Bdomain sequence to a B domain deleted FVIII, optimally 226 amino acidsand retaining 6 sites for N-linked glycosylation, to improve secretion.McIntosh et al. (2013) replaced the 226 amino-acid spacer of Miao et al.with a 17 amino-acid peptide in which six glycosylation triplets fromthe B-domain were juxtaposed. Yet, production was still not sufficientfor therapeutic purposes.

Non-viral vectors typically rely on a plasmid-based gene deliverysystem, where only the naked DNA is delivered, potentially inconjunction with physicochemical methods that facilitate transfection.Consequently, the non-viral approach may be less immunogenic andpotentially safer than viral vectors, though innate immune response maystill occur. The non-viral gene transfer method is simple, but theefficiency is generally low compared to most viral vector-mediated genetransfer approaches. Efficient in vivo gene delivery of non-viralvectors remains a bottleneck. Typically, for hepatic gene delivery,plasmids are administered by hydrodynamic injection. In this case, ahydrodynamic pressure is generated by rapid injection of a large volumeof DNA solution into the circulation, in order to deliver the gene ofinterest in the liver (Miao et al., 2000). Efforts are being made toadapt hydrodynamic injection towards a clinically relevant modality byreducing the volume of injection along with maintaining localizedhydrodynamic pressure for gene transfer. Alternative approaches based ontargetable nanoparticles are being explored to achieve target specificdelivery of FIX into hepatocytes. Expression could be prolonged byremoving bacterial backbone sequences which interfere with long termexpression (i.e. mini-circle DNA). Finally, to increase the stability ofFIX expression after non-viral transfection, transposons could be usedthat result in stable genomic transgene integration. We and others haveshown that transposons could be used to obtain stable clotting factorexpression following in vivo gene therapy (Yant et al., 2000; Mates,Chuah et al., 2009, VandenDriessche et al., 2009; Kren et al.,2009;Ohlfest et al., 2004).

An exemplary state of the art vector for liver-specific expression ofFIX is described in WO 2009/130208 and is composed of a single-strandedAAV vector that contains the TTR/Serp regulatory sequences driving afactor cDNA. A FIX first intron was included in the vector, togetherwith a poly-adenylation signal. Using said improved vector yielded about25-30% stable circulating factor IX.

In order to translate viral-vector based gene therapy for hemophilia tothe clinic, the safety concerns associated with administering largevector doses to the liver and the need for manufacturing large amountsof clinical-grade vector must be addressed. Increasing the potency(efficacy per dose) of gene transfer vectors is crucial towardsachieving these goals. It would allow using lower doses to obtaintherapeutic benefit, thus reducing potential toxicities and immuneactivation associated with in vivo administration, and easingmanufacturing needs.

One way to increase potency is to engineer the transgene sequence itselfto maximize expression and biological activity per vector copy. We haveshown that FIX transgenes optimized for codon usage and carrying anR338L amino acid substitution associated with clotting hyperactivity andthrombophilia (Simioni et al., 2009), increase the efficacy of genetherapy using lentiviral vector up to 15-fold in hemophilia B mice,without detectable adverse effects, substantially reducing the doserequirement for reaching therapeutic efficacy and thus facilitatingfuture scale up and its clinical translation (Cantore et al., 2012, Nairet al., 2014).

Also codon optimization of human factor VIII cDNAs leads to high-levelexpression. Significantly greater levels (up to a 44-fold increase andin excess of 200% normal human levels) of active FVIII protein weredetected in the plasma of neonatal hemophilia A mice transduced withlentiviral vector expressing FVIII from a codon-optimized cDNA sequence,thereby successfully correcting the disease model (Ward et al., 2011).

In WO 2014/064277 expression vectors are described which combine therobust Serpin enhancer with codon-optimized transgenes encoding FIX orFVIII, resulting in increased liver-specific expression of FIX andFVIII, respectively.

It is an object of the present invention to further increase theefficiency and safety of liver-directed gene therapy for hemophilia Aand B.

SUMMARY OF THE INVENTION

It is an object of the present invention to increase the efficiency andsafety of liver-directed gene therapy for hemophilia B. The aboveobjective is accomplished by providing a nucleic acid expressioncassette and a vector, either a viral vector, in particular an AAV-basedvector, or a non-viral vector, comprising specific regulatory elementsthat enhance liver-directed gene expression, while retaining tissuespecificity, in conjunction with the use of a transgene, preferably acodon-optimized transgene, encoding human FIX, preferably human FIXcontaining a hyper-activating mutation.

The resulting vector and nucleic acid expression cassette result inunexpectedly high expression levels of FIX in the liver, due to itsunique combination of regulatory elements. The combined effect of theseelements could not have been predicted. Previously, we reported on a newregulatory element that in combination with other vector elementsrepresented a more than 20-fold increase in FIX levels (cf.WO2014/064277). In the present invention, it is shown that combining 3copies of the serpin enhancer with the known natural TTR enhancerfurther increases FIX expression by 6 to 10 fold. This increase in hFIXactivity was shown to be synergistic. It is another object of thepresent invention to increase the efficiency and safety ofliver-directed gene therapy for hemophilia A. As shown in theexperimental section, this objective is accomplished by providing avector, either a viral vector, in particular an AAV-based vector, or anon-viral vector, comprising a nucleic acid expression cassette withspecific regulatory elements that enhance liver-directed geneexpression, while retaining tissue specificity, in conjunction with theuse of a codon-optimized human FVIII construct, in particular acodon-optimized B domain deleted FVIII construct driven from a minimaltransthyretin promoter. The resulting AAV-based vector and nucleic acidexpression cassette resulted in unprecedented, supra-physiologic FVIIIexpression levels (cf. WO2014/064277). The inventors now demonstratedthat the specific combination of three copies of the Serpin enhancerwith the natural TTR enhancer, and the codon-optimized B domain deletedFVIII transgene or the codon optimized padua mutant FIX transgene drivenfrom a minimal transthyretin promoter provides for a synergistic effecton FVIII or FIX expression levels compared to expression cassettescontaining the natural TTR enhancer and the codon-optimized B domaindeleted FVIII transgene or the codon optimized padua mutant FIXtransgene driven from a minimal transthyretin promoter.

The combination of the triple repeat of the Serpin enhancer defined bySEQ ID NO:5 and the transthyretin enhancer defined by SEQ ID NO:12 hasbeen shown to be unexpectedly potent in increasing expression of atransgene operably linked to it. Said regulatory element is defined bySEQ ID NO:13. Said regulatory element can further be combined with thetransthyretin minimal promotor as defined by SEQ ID NO.6. This creates acombination of 3x the SerpEnh (3× SEQ ID NO.5, e.g. such as in SEQ IDNO.11) with the TTRe and TTRm nucleic acid sequence e.g. as defined bySEQ ID NO.69. For example, such a construct results in a regulatoryelement as defined by SEQ ID NO:58, which has been tested to increasethe expression of both FVIII and FIX transgenes as shown herein.

The invention therefore provides the following aspects:

Aspect 1. A nucleic acid expression cassette comprising a triple repeat,preferably a tandem repeat, of a liver-specific nucleic acid regulatoryelement comprising or consisting of the nucleic acid fragment defined bySEQ ID NO:5 or a sequence having at least 95% identity to said sequence,preferably a liver-specific nucleic acid regulatory element of 150nucleotides or less, comprising or consisting of the nucleic acidfragment defined by SEQ ID NO:5 or a sequence having at least 95%identity to said sequence, more preferably the regulatory element (3×SERP) as defined by SEQ ID NO:11, operably linked to a promoter and atransgene, preferably a codon-optimized transgene, wherein the promoteris a liver-specific promoter.

Preferably said promoter is derived from the transthyretin (TTR)promoter, more preferably said promoter is the minimal TTR promotor(TTRm) as defined by SEQ ID NO:6.

In another preferred embodiment, said liver-specific promoter is derivedfrom the AAT promoter, e.g. as defined by SEQ ID NO.64.

In further embodiments, the liver-specific promotor is selected from thegroup comprising: the transthyretin (TTR) promoter or TTR-minimalpromoter (TTRm), the alpha 1-antitrypsin (AAT) promoter, the albuminpromotor (ALB) or minimal albumin promoter (ALBm), the apolipoprotein Al(APOA1) promoter, the complement factor B (CFB) promoter, theketohexokinase (KHK) promoter, the hemopexin (HPX) promoter, thenicotinamide N-methyltransferase (NNMT) promoter, the (liver)carboxylesterase 1 (CES1) promoter, the protein C (PROC) promoter, theapolipoprotein C3 (APOC3) promoter, the mannan-binding lectin serineprotease 2 (MASP2) promoter, the hepcidin antimicrobial peptide (HAMP)promoter, or the serpin peptidase inhibitor, clade C (antithrombin),member 1 (SERPINC1) promoter.

Aspect 2. The nucleic acid expression cassette according to aspect 1,further comprising a nucleic acid regulatory element comprising orconsisting of the nucleic acid fragment defined by SEQ ID NO:12 (TTRe),or a sequence having at least 95% identity to said sequence, preferablya nucleic acid regulatory element of 150 nucleotides or less comprisingor consisting of the nucleic acid fragment defined by SEQ ID NO:12, or asequence having at least 95% identity to said sequence. In a preferredembodiment, the combination of the TTRe and TTRm nucleic acid modules isdefined by SEQ ID NO.69.

Aspect 3, The nucleic acid expression cassette according to aspect 2,comprising the nucleic acid regulatory element as defined by SEQ IDNO:13, preferably the nucleic acid regulatory element as defined by SEQID NO:57, operably linked to a promotor and a transgene, or the nucleicacid regulatory element as defined by SEQ ID NO:58, operably linked to atransgene.

Aspect 4. The nucleic acid expression cassette according to any one ofaspects 1 to 3, wherein said transgene encodes for coagulation factorVIII or coagulation factor IX.

Aspect 5. The nucleic acid expression cassette according to aspect 4,wherein said coagulation factor VIII has a deletion of the B domain.

Aspect 6. The nucleic acid expression cassette according to aspect 5,wherein said B domain of said FVIII is replaced by a linker defined bySEQ ID NO:59.

Aspect 7. The nucleic acid expression cassette according to any one ofaspects 4 to 6, wherein said transgene encoding for coagulation factorVIII is defined by SEQ ID NO: 18.

Aspect 8. The nucleic acid expression cassette according to aspect 4,wherein said coagulation factor IX contains a hyper-activating mutation.

Aspect 9. The nucleic acid expression cassette according to aspect 8,wherein said hyper-activating mutation in coagulation factor IXcorresponds to an R338L amino acid substitution.

Aspect 10. The nucleic acid expression cassette according to any one ofaspects 4, 8 or 9, wherein said transgene encoding for coagulationfactor IX is defined by SEQ ID NO:9.

Aspect 11. The nucleic acid expression cassette according to any one ofaspects 1 to 10, wherein said promoter is a liver-specific promoter,preferably a promoter derived from the transthyretin (TTR) promoter,preferably the minimal TTR promotor as defined by SEQ ID NO:6.

Aspect 12. The nucleic acid expression cassette according to any one ofaspects 1 to 11, further comprising a minute virus of mouse (MVM)intron, preferably the MVM intron as defined by SEQ ID NO:8.

Aspect 13. The nucleic acid expression cassette according to any one ofaspects 1 to 12, further comprising a transcriptional termination signalderived from the bovine growth hormone polyadenylation signal (BGHpA),preferably the BGHpA as defined by SEQ ID NO:10, or derived from theSimian virus 40 polyadenylation signal (SV40pA), preferably the SV40pAas defined by SEQ ID NO:19, or a synthetic polyadenylation signal,preferably the synthetic polyadenylation signal as defined by SEQ ID NO:56.

Aspect 14. A vector comprising the nucleic acid expression cassetteaccording to any one of aspects 1 to 13.

Aspect 15. The vector according to aspect 14, wherein said vector is aviral vector.

Aspect 16. The vector according to aspect 15, wherein said vector isderived from an adeno-associated virus (AAV).

Aspect 17. The vector according to aspect 16, wherein said vector is asingle-stranded AAV vector, preferably a single-stranded AAV serotype 8vector.

Aspect 18. The vector according to any one of aspects 14 to 17, definedby SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ IDNO:23, preferably SEQ ID NO:22 or SEQ ID NO:23, more preferably SEQ IDNO:22.

Aspect 19. The vector according to aspect 16, wherein said vector is aself-complementary AAV vector, preferably a self-complementary AAVserotype 9 vector.

Aspect 20. The vector according to any one of aspects 14 to 16, or 19,defined by SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:25, preferably SEQ IDNO:4 or SEQ ID NO:25, more preferably SEQ ID NO:4, or the vectoraccording to any one of aspects 14 to 16, or 19, defined by SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:68, preferably SEQ ID NO:65 or 68, morepreferably SEQ ID NO:65.Aspect 21. The vector according to aspect 14,wherein said vector is a non-viral vector, such as a transposon-basedvector (e.g. a PiggyBac(PB)-based vector or a Sleeping Beauty(SB)-basedvector).

Aspect 22. A method to obtain levels of factor VIII in plasma equal toor higher than the therapeutic threshold concentration of 10 plasma in asubject, comprising the transduction or transfection of the vectoraccording to any one of aspects 14 to 18, or 21 into a subject.

Aspect 23. The method according to aspect 22, wherein the transductionof the vector according to any one of aspects 14 to 18, or 21 into thesubject is done at a dose lower than 2.5×10¹¹ vg/kg.

Aspect 24. A method to obtain levels of factor IX in plasma equal to orhigher than the therapeutic threshold concentration of 10 mU/ml plasmain a subject, comprising the transduction or transfection of the vectoraccording to any one of aspects 14 to 16, or 19 to 21 into a subject.

Aspect 25. The method according to aspect 24, wherein the transductionof the vector according to any one of aspects 14 to 16, or 19 to 21 intothe subject is done at a dose lower than 2×10¹¹ vg/kg.

Aspect 26. The method according to any one of aspects 22 to 25, whereinsaid transduction or transfection is by intravenous administration.

Aspect 27. The method according to any one of aspects 22 to 26, whereinsaid subject is a mammalian subject, preferably a human subject.

Aspect 28. A method for treating hemophilia A in a mammalian subject,comprising performing the method according to any one of aspects 22, 23,26 or 27.

Aspect 29. The use of the vector according to any one of aspects 14 to18, or 21 for the manufacture of a medicament to treat hemophilia A.

Aspect 30. The vector according to any one of aspects 14 to 18, or 21for use in the treatment of hemophilia A.

Aspect 31. A method for treating hemophilia B in a mammalian subject,comprising performing the method according to any one of aspects 24 to27.

Aspect 32. The use of the vector according to any one of aspects 14 to16, or 19 to 21 for the manufacture of a medicament to treat hemophiliaB.

Aspect 33. The vector according to any one of aspects 14 to 16, 19 to 21for use in the treatment of hemophilia B.

Aspect 34. A pharmaceutical composition comprising a vector according toany one of aspects 14 to 18, or 21 and a pharmaceutically acceptablecarrier, optionally further comprising an active ingredient for treatinghemophilia A.

Aspect 35. The pharmaceutical composition according to aspect 34 for usein treating hemophilia A.

Aspect 36. The pharmaceutical composition for use according to aspect35, or the vector for use according to aspect 30, wherein said treatmentresults in levels of factor VIII in plasma of the treated subject thatare equal to or higher than the therapeutic threshold concentration of10 mU/ml plasma in a subject.

Aspect 37. The pharmaceutical composition for use according to any oneof aspects 35 or 35, or the vector for use according to any one ofaspects 30 or 36, wherein said treatment comprises the transduction ofthe vector according to any one of aspects 14 to 18 into the subject ata dose lower than or equal than 2.5×10¹¹ vg/kg.

Aspect 38. A pharmaceutical composition comprising a vector according toany one of aspects 14 to 16, 19 to 21 and a pharmaceutically acceptablecarrier, optionally further comprising an active ingredient for treatinghemophilia B.

Aspect 39. The pharmaceutical composition according to aspect 38, foruse in treating hemophilia B.

Aspect 40. The pharmaceutical composition for use according to aspect39, or the vector for use according to aspect 33, wherein said treatmentresults in levels of factor IX in plasma of the treated subject that areequal to or higher than the therapeutic threshold concentration of 10mU/ml plasma in a subject, preferably equal to or higher than thetherapeutic concentration of 50 mU/ml plasma in a subject, morepreferably equal to or higher than the therapeutic concentration of 100mU/nnl plasma in a subject, even more preferably equal to or higher thanthe therapeutic concentration of 150 mU/ml plasma in a subject and evenmore preferably equal to or higher than the therapeutic concentration of200 mU/ml plasma in a subject.

Aspect 41. The pharmaceutical composition for use according to aspect 39or 40, or the vector for use according to aspect 33 or 40, wherein saidtreatment comprises the transduction of the vector according to any oneof aspects 14 to 16, 19 or 20 into the subject at a dose lower than orequal than 2×10¹² vg/kg, preferably at a dose lower than or equal than6×10¹¹ vg/kg, more preferably ata dose lower than or equal than2×10¹¹vg/kg.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is illustrated by the following figures which areto be considered for illustrative purposes only and in no way limit theinvention to the embodiments disclosed therein:

FIG. 1: Design of the self-complementary (sc), double-strandedadeno-associated viral (AAV) vectorsAAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA (2510 bp). FIG. 1A:AAVsc-3×SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA (2683 bp). FIG. 1B:AAVsc-TTREnh-TTRm-MVM-co-FIX-R338L-BGHpA (2540 bp). FIG. 1C:AAVsc-3×SerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L-BGHpA (2760 bp). FIG. 1D:and AAVsc-3×SerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L-Synt.pA (2519 bp). FIG.1E: The minimal transthyretin promoter (TTRm) is driving the expressionof the codon-optimized human factor IX (co-FIX-R338L) containing thehyper-activating, thrombophilic FIX mutation (R338L). Either the nativeTTR enhancer (TTRe), the Serpin enhancer (SERP), a triplet of the Serpinenhancer (3×SERP), or a combination of a triplet of the Serpin enhancer(3×SERP) and the native TTR enhancer (TTRe) is cloned upstream of TTRm.The minute virus of mouse intron (MVM), the bovine growth hormonepolyadenylation site (BGHpA) or a synthetic polyadenylation site(Synt.pA), and the inverted terminal repeats (ITR) are indicated. Thesequences flanking/linking the different elements are indicated. Theindicated vector sizes includes both ITR's.

FIG. 2: Design of the single-stranded (ss) adeno-associated viral (AAV)vectors AAVss-TTRm-MVM-co-hFVIII-ΔB-Sv40pA (5160 bp). FIG. 2A:AAVss-SerpEnh-TTRm-MVM-co-hFVIII-ΔB-Sv40pA (5252 bp). FIG. B:AAVss-3×SerpEnh-TTRm-MVM-co-hFVIII-ΔB-Sv40pA (5410 bp). FIG. C:AAVss-TTREnh-TTRm-MVM-co-hFVIII-ΔB-Sv40pA (5272 bp). FIG. D:AAVss-3×SerpEnh-TTRm-MVM-co-hFVIII-ΔB-Synt.pA (5325 bp). FIG. E:AAVss-3×SerpEnh-TTRm-co-hFVIII-AB-Synt .pA (5217 bp). FIG. F:AAVss-3×SerpEnh-TTREnh-TTRm-MVM-co-hFVIII-AB-Sv40pA (5493 bp). FIG. G:AAVss-3×SerpEnh-TTREnh-TTRm-co-hFVIII-ΔB-Synt.pA (5307 bp). FIG. H: andAAVss-TTREnh-TTRm-co-hFVIII-AB-Synt.pA (5083 bp). FIG. I: The minimaltransthyretin promoter (TTRm) is driving the expression of the humancodon-optimized B-domain deleted factor VIII (cohFVIIIdeltaB). Thenative TTR enhancer (TTRe), the Serpin enhancer (SERP), a triplet of theSerpin enhancer (3×SERP), or a combination of a triplet of the Serpinenhancer (3×SERP) and the native TTR enhancer (TTRe) may be clonedupstream of TTRm. The minute virus of mouse intron (MVM), the SimianVirus 40 (SV40) polyadenylation site (SV40pA) or a syntheticpolyadenylation site (Synt.pA), and the inverted terminal repeats (ITR)are indicated. The sequences flanking/linking the different elements areindicated. The indicated vector sizes include both ITR's.

FIG. 3: FIX protein levels upon transduction of the describedSC-vectors. FIG. 3A: the protein expression levels achieved upontransduction with 1×10e9 vg/mouse (Low Dose) for the different vectorsystems described in FIG. 1. FIG. 3B: the protein expression levelsachieved upon transduction with 5×10e9 vg/mouse (High Dose) for thedifferent vector systems described in FIG. 1.

FIG. 4: FVIII protein levels upon transduction of the describedSS-vectors. FIG. 4A: the protein expression levels achieved upontransduction with 1×10e9 vg/mouse (Low Dose) for the different vectorsystems described in FIG. 2. FIG. 4B: the protein expression levelsachieved upon transduction with 5×10e9 vg/mouse (High Dose) for thedifferent vector systems described in FIG. 2.

FIG. 5: Plasmid map of the pAAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpAvector

FIG. 6: Plasmid map of the pAAVsc-3×SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpAvector

FIG. 7: Plasmid map of the pAAVsc-TTRe-TTRm-MVM-co-FIX-R338L-BGHpAvector

FIG. 8: Plasmid map of thepAAVsc-3×SerpEnh-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA vector

FIG. 9: Plasmid map of the pAAVss-TTRm-MVM-coFVIIIdeltaB-Sv40pA vector

FIG. 10: Plasmid map of the pAAVss-SerpEnh-TTRm-MVM-coFVIIIdeltaB-Sv40pAvector

FIG. 11: Plasmid map of thepAAVss-3×SerpEnh-TTRm-MVM-coFVIIIdeltaB-Sv40pA vector

FIG. 12: Plasmid map of the pAAVss-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pAvector

FIG. 13: Nucleotide sequence ofpAAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:1);pAAVsc-3×SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:2);pAAVsc-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:3);pAAVsc-3×SerpEnh-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:4); theSerpin enhancer (SerpEnh) (SEQ ID NO:5); the minimal transthyretin(TTRm) promoter (SEQ ID NO:6); the 5′ untranslated region (UTR) of TTRm(TTRm5′UTR) (SEQ ID NO:7); the Minute Virus of Mouse (MVM) intron (SEQID NO:8); the codon-optimized transgene encoding human FIX Padua mutant(Co-FIX-R338L) (SEQ ID NO:9); the Bovine Growth Hormone polyadenylationsignal (BGHpA) (SEQ ID NO:10); triple tandem repeat of the Serpinenhancer (3×SERP) (SEQ ID NO:11, the nucleotide linking the repeats isindicated in bold); the transthyretin enhancer (TTRe) (SEQ ID NO:12); atriple tandem repeat of the Serpin enhancer (underlined) linked to thetransthyretin enhancer (italics) (3×SERP-Flank-TTRe) (SEQ ID NO:13);pAAVsc-3×SerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L-Synt.pA (SEQ ID NO:25);Flank-3×SERP-Flank-TTRe (SEQ ID NO:26); Flank-3×SERP-Flank-TTRe-Flank(SEQ ID NO:27); Flank-3×SERP-Flank-TTRe-Flank-TTRm (SEQ ID NO:28);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank (SEQ ID NO:29);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-MVM (SEQ ID NO:30);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank (SEQ ID NO:31);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L (SEQ IDNO:32),Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Flank(SEQ ID NO:33);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Flank-BGHpA(SEQ ID NO:34);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Flank-BGHpA-Flank(SEQ ID NO:35);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Flank-Synt.pA(SEQ ID NO:36);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Flank-Synt.pA-Flank(SEQ ID NO:37).

FIG. 14: Nucleotide sequence of pAAVss-TTRm-MVM-coFV1IldeltaB-Sv40pA(SEQ ID NO:14); pAAVss-SerpEnh-TTRm-MVM- coFVIIIdeltaB-Sv40pA (SEQ IDNO:15); pAAVss-3×SerpEnh-TTRm-MVM- coFVIIIdeltaB-Sv40pA (SEQ ID NO:16);pAAVss-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:17);codon-optimized transgene encoding B domain deleted human factor VIII(coFVIIIdeltaB) (SEQ ID NO:18); Simian virus 40 polyadenylation signal(SV40polyA) (SEQ ID NO:19);pAAVss-3×SerpEnh-TTRm-MVM-coFVIIIdeltaB-Synt.pA (SEQ ID NO:20);pAAVss-3×SerpEnh-TTRm-coFV1IldeltaB-Synt.pA (SEQ ID NO:21);pAAVss-3×SerpEnh-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:22);pAAVss-3×SerpEnh-TTRe-TTRm-coFVIIIdeltaB-Synt.pA (SEQ ID NO:23);pAAVss-TTRe-TTRnn-coFVIIIdeltaB-Synt. pA (SEQ ID NO:24);Flank-3×SERP-F/ank (SEQ ID NO:38); Flank-3×SERP-Flank-TTRe (SEQ IDNO:39); Flank-3×SERP-Flank-TTRe-Flank (SEQ ID NO:40);Flank-3×SERP-Flank-TTRe-Flank-TTRm (SEQ ID NO:41);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank (SEQ ID NO:42);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB (SEQ ID NO:43);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank (SEQ IDNO:44);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank-SV40pA (SEQID NO:45);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank-SV40pA-Flank(SEQ ID NO:46); Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank-Synt.pA (SEQ ID NO:47);Flank-3×SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank-SyntpA-Flank(SEQ ID NO:48); Flank-3×SERP-Flank-TTRe-Flank-TTRm-MVM-Flank (SEQ IDNO:49); Flank-3×SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB (SEQID NO:50);Flank-3×SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank (SEQ IDNO:51);Flank-3×SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank-SV40pA(SEQ ID NO:52);Flank-3×SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank-SV40pA-Flank(SEQ ID NO:53);Flank-3×SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank-Synt.pA(SEQ ID NO:54);Flank-3×SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank-Synt.pA-Flank(SEQ ID NO:55); synthetic polyadenylation signal (Synt.pA) (SEQ IDNO:56); 3×SERP-Flank-TTRe-Flank (SEQ ID NO:57);3×SERP-Flank-TTRe-Flank-TTRm (SEQ ID NO:58).

FIG. 15: FVIII protein levels in C57BL6 mice injected with thepAAVss-3×SerpEnh-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA andpAAVss-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA plasmids as described inFIG. 2. C57BL6 mice were injected with 300 ng of the respectiveplasmids. FVIII protein levels were measured by ELISA in plasma samplescollected on day 1 (black bar) and day 2 (white bar) post injection.

FIG. 16: Plasmid map of thepAAVsc-3×SerpEnh-TTRe-TTRm-MVM-co-FIX-R338L-Synt.pA vector

FIG. 17: Plasmid map of thepAAVss-3×SerpEnh-TTRm-MVM-coFVIIIdeltaB-Synt.pA vector

FIG. 18: Plasmid map of the pAAVss-3×SerpEnh-TTRm-coFVIIIdeltaB-Synt.pAvector

FIG. 19: Plasmid map of thepAAVss-3×SerpEnh-TTRe-TTRm-MVM-coFV1IldeltaB-Sv40pA vector

FIG. 20: Plasmid map of thepAAVss-3×SerpEnh-TTRe-TTRm-coFVIIIdeltaB-Synt.pA vector

FIG. 21: Plasmid map of the pAAVss-TTRe-TTRm-coFVIIIdeltaB-Synt.pAvector.

FIG. 22: Design of comparative example of self-complementary (sc),double-stranded adeno-associated viral (AAV) vectors expressing co-FVIIIdelta-B. The following vectors were compared for FVIII expressionlevels: pAAVss-TTRm-MVM-coFV1IldeltaB-Sv40pA (SEQ ID NO. 14);pAAVss-3×SerpEnh-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:16);pAAVss-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:17) andpAAVss-3×SerpEnh-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:22).

FIG. 23: Design of comparative example of self-complementary (sc),double-stranded adeno-associated viral (AAV) vectors expressing co-FIX.The following vectors were compared for FIX expression:pAAVss-3×SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:65);pAAVss-3×SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:66);pAAVss-3×SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:67); andpAAVss-3×SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:68).

FIG. 24: Nucleotide sequence of AAT promotor (SEQ ID 64);pAAVss-3×SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:65);pAAVss-3×SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:66);pAAVss-3×SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:67); andpAAVss-3×SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:68).

FIG. 25: FIG. 25A: Graph depicting the effect of the 3×Serp enhancer onthe TTRenh-TTRm promoter in regulating the FVIII expression in C57BL6mice injected with 300 ng of respective plasmids. The FVIII expressionprofile was tested using the ELISA, in the plasma samples collectedDay 1. The presence of 3× Serp enhancer seems to elevate the FVIIIexpression by 8 fold. FIG. 25B: Graph comparing different constructstested on FVIII expression as outlined in example 2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described with respect to particularembodiments and with reference to certain drawings but the invention isnot limited thereto but only by the claims. Any reference signs in theclaims shall not be construed as limiting the scope. The drawingsdescribed are only schematic and are non-limiting. In the drawings, thesize of some of the elements may be exaggerated and not drawn on scalefor illustrative purposes.

Where the term “comprising” is used in the present description andclaims, it does not exclude other elements or steps. The term“comprising” also encompasses the more specific embodiments defined as“consisting of” and “consisting essentially of”.

Where an indefinite or definite article is used when referring to asingular noun e.g. “a” or “an”, “the”, this includes a plural of thatnoun unless something else is specifically stated. Furthermore, theterms first, second, third and the like in the description and in theclaims, are used for distinguishing between similar elements and notnecessarily for describing a sequential or chronological order.

It is to be understood that the terms so used are interchangeable underappropriate circumstances and that the embodiments of the inventiondescribed herein are capable of operation in other sequences thandescribed or illustrated herein.

The following terms or definitions are provided to aid in theunderstanding of the invention. Unless specifically defined herein, allterms used herein have the same meaning as they would to one skilled inthe art of the present invention. Practitioners are particularlydirected to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubelet al., Current Protocols in Molecular Biology (Supplement 47), JohnWiley & Sons, New York (1999), for definitions and terms of the art.

The definitions provided herein should not be construed to have a scopeless than understood by a person of ordinary skill in the art.

The invention relates to nucleic acid expression cassettes andexpression vectors comprising said nucleic acid expression cassettes forenhancing liver-specific expression of a (trans)gene.

As used herein, the term “nucleic acid expression cassette” refers tonucleic acid molecules that include one or more transcriptional controlelements (such as, but not limited to promoters, enhancers and/orregulatory elements, polyadenylation sequences, and introns) that direct(trans)gene expression in one or more desired cell types, tissues ororgans. Typically, the nucleic acid expression cassettes describedherein will contain the FIX transgene or the FVIII transgene as definedherein.

The invention provides nucleic acid expression cassettes comprising oneor more liver-specific nucleic acid regulatory elements, operably linkedto a promoter and a transgene.

The term “operably linked” as used herein refers to the arrangement ofvarious nucleic acid molecule elements relative to each such that theelements are functionally connected and are able to interact with eachother. Such elements may include, without limitation, a promoter, anenhancer and/or a regulatory element, a polyadenylation sequence, one ormore introns and/or exons, and a coding sequence of a gene of interestto be expressed (i.e., the transgene). The nucleic acid sequenceelements, when properly oriented or operably linked, act together tomodulate the activity of one another, and ultimately may affect thelevel of expression of the transgene. By modulate is meant increasing,decreasing, or maintaining the level of activity of a particularelement. The position of each element relative to other elements may beexpressed in terms of the 5′ terminus and the 3′ terminus of eachelement, and the distance between any particular elements may bereferenced by the number of intervening nucleotides, or base pairs,between the elements.

A “regulatory element” as used herein refers to transcriptional controlelements, in particular non-coding cis-acting transcriptional controlelements, capable of regulating and/or controlling transcription of agene, in particular tissue-specific transcription of a gene. Regulatoryelements comprise at least one transcription factor binding site (TFBS),more in particular at least one binding site for a tissue-specifictranscription factor, most particularly at least one binding site for aliver-specific transcription factor. Typically, regulatory elements asused herein increase or enhance promoter-driven gene expression whencompared to the transcription of the gene from the promoter alone,without the regulatory elements. Thus, regulatory elements particularlycomprise enhancer sequences, although it is to be understood that theregulatory elements enhancing transcription are not limited to typicalfar upstream enhancer sequences, but may occur at any distance of thegene they regulate. Indeed, it is known in the art that sequencesregulating transcription may be situated either upstream (e.g. in thepromoter region) or downstream (e.g. in the 3′UTR) of the gene theyregulate in vivo, and may be located in the immediate vicinity of thegene or further away. Of note, although regulatory elements as disclosedherein typically are naturally occurring sequences, combinations of(parts of) such regulatory elements or several copies of a regulatoryelement, i.e. non-naturally occurring sequences, are themselves alsoenvisaged as regulatory element. Regulatory elements as used herein maybe part of a larger sequence involved in transcriptional control, e.g.part of a promoter sequence. However, regulatory elements alone aretypically not sufficient to initiate transcription, but require apromoter to this end.

The one or more regulatory elements contained in the nucleic acidexpression cassettes and vectors disclosed herein are preferablyliver-specific. Non-limiting examples of liver-specific regulatoryelements are disclosed in WO 2009/130208, which is specificallyincorporated by reference herein. Another example of a liver-specificregulatory element is a regulatory element derived from thetransthyretin (TTR) gene, such as the regulatory element defined by SEQID NO:12, also referred to herein as “TTRe” or “TTREnh”(Wu et al.,2008). ‘Liver-specific expression’, as used in the application, refersto the preferential or predominant expression of a (trans)gene (as RNAand/or polypeptide) in the liver as compared to other tissues. Accordingto particular embodiments, at least 50% of the (trans)gene expressionoccurs within the liver. According to more particular embodiments, atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 99% or 100% of the(trans)gene expression occurs within the liver. According to aparticular embodiment, liver-specific expression entails that there isno ‘leakage’ of expressed gene product to other organs, such as spleen,muscle, heart and/or lung. The same applies mutatis mutandis forhepatocyte-specific expression, which may be considered as a particularform of liver-specific expression. Throughout the application, whereliver-specific is mentioned in the context of expression,hepatocyte-specific expression is also explicitly envisaged. Similarly,where tissue-specific expression is used in the application, cell-typespecific expression of the cell type(s) predominantly making up thetissue is also envisaged.

Preferably, the one or more regulatory element in the nucleic acidexpression cassettes and vectors disclosed herein is fully functionalwhile being only of limited length. This allows its use in vectors ornucleic acid expression cassettes without unduly restricting theirpayload capacity. Accordingly, in embodiments, the one or moreregulatory element in the expression cassettes and vectors disclosedherein is a nucleic acid of 1000 nucleotides or less, 800 nucleotides orless, or 600 nucleotides or less, preferably 400 nucleotides or less,such as 300 nucleotides or less, 200 nucleotides or less, 150nucleotides or less, or 100 nucleotides or less (i.e. the nucleic acidregulatory element has a maximal length of 1000 nucleotides, 800nucleotides, 600 nucleotides, 400 nucleotides, 300 nucleotides, 200nucleotides, 150 nucleotides, or 100 nucleotides).

However, it is to be understood that the disclosed nucleic acidregulatory elements retain regulatory activity (i.e. with regard tospecificity and/or activity of transcription) and thus they particularlyhave a minimum length of 20 nucleotides, 25 nucleotides, 30 nucleotides,35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55nucleotides, 60 nucleotides, 65 nucleotides, or 70 nucleotides. Inpreferred embodiments, the one or more regulatory element in the nucleicacid expression cassettes and vectors disclosed herein comprises asequence from SERPINA1 regulatory elements, i.e. regulatory elementsthat control expression of the SERPINA1 gene in vivo. Said regulatoryelement preferably comprises, consists essentially of or consists of thesequence as defined in SEQ ID NO:5, a sequence having at least 85%,preferably at least 90%, more preferably at least 95%, such as 96%, 97%,98% or 99%, identity to said sequence, or a functional fragment thereof.Also preferably, said regulatory element has a maximal length of 150nucleotides or less, preferably 100 nucleotides or less, and comprises,consists essentially of or consists of the sequence as defined in SEQ IDNO:5, a sequence having at least 85%, preferably at least 90%, morepreferably at least 95%, such as 96%, 97%, 98% or 99%, identity to saidsequence, or a functional fragment thereof. The liver-specific nucleicacid regulatory element consisting of SEQ ID NO:5 is herein referred toas “the Serpin enhancer”, “SerpEnh”, or “Serp”.

In particularly preferred embodiments, the nucleic acid expressioncassettes and vectors disclosed herein comprise two or more, such astwo, three, four, five or six, preferably three, (tandem) repeats of aliver-specific regulatory element comprising, consisting essentially ofor consisting of SEQ ID NO:5, or a sequence having at least 85%,preferably at least 90%, more preferably at least 95%, such as 96%, 97%,98% or 99%, identity to said sequence, more preferably a liver-specificregulatory element of 150 nucleotides or less, preferably 100nucleotides or less, more preferably 80 nucleotides or less, comprising,consisting essentially of or consisting of SEQ ID NO:5, or a sequencehaving at least 85%, preferably at least 90%, more preferably at least95%, such as 96%, 97%, 98% or 99%, identity to said sequence. Apreferred nucleic acid regulatory element comprising three tandemrepeats of SEQ ID NO:5 is herein referred to as “3×Serp” and is definedby SEQ ID NO:11.

In further embodiments, the nucleic acid expression cassettes andvectors disclosed herein comprise two or more, such as two, three, four,five or six, more preferably three, (tandem) repeats of a liver-specificregulatory element comprising, consisting essentially of or consistingof SEQ ID NO:5, or a sequence having at least 85%, preferably at least90%, more preferably at least 95%, such as 96%, 97%, 98% or 99%,identity to said sequence, preferably a liver-specific regulatoryelement of 150 nucleotides or less, preferably 100 nucleotides or less,more preferably 80 nucleotides or less, comprising, consistingessentially of or consisting of SEQ ID NO:5, or a sequence having atleast 85%, preferably at least 90%, more preferably at least 95%, suchas 96%, 97%, 98% or 99%, identity to said sequence; and a regulatoryelement comprising, consisting essentially of or consisting of SEQ IDNO:12, preferably a regulatory element of 150 nucleotides or less,preferably 120 nucleotides or less, comprising, consisting essentiallyof or consisting of SEQ ID NO:12. A preferred liver-specific nucleicacid regulatory element comprising three tandem repeats of SEQ ID NO:5,and SEQ ID NO:12, is herein referred to as “3×Serp-flank-TTRe” and isdefined by SEQ ID NO:13 A further preferred liver-specific nucleic acidregulatory element comprising three tandem repeats of SEQ ID NO:5, andSEQ ID NO:12, is herein referred to as “3×Serp-flank-TTRe-flank” and isdefined by SEQ

ID NO:57. Preferably, the liver-specific nucleic acid regulatory elementin the nucleic acid expression cassettes and vectors disclosed hereincomprises three tandem repeats of SEQ ID NO:5, and SEQ ID NO:12; morepreferably SEQ ID NO:13; even more preferably SEQ ID NO:57, such as SEQID NO:27 or SEQ ID NO:39. It has been shown herein that said specificcombinations of liver-specific regulatory elements resulted inunexpectedly enhanced liver-specific expression of the transgene, inparticular the FIX transgene or FVIII transgene described herein,operably linked thereto.

In further embodiments, said nucleic acid expression cassettes andvectors disclosed herein comprise three tandem repeats of SEQ ID NO:5(such as SEQ ID NO.13), and a further enhancer element TTRe defined bySEQ ID NO:12, for example as defined by SEQ ID NO:13, in combinationwith a liver-specific promotor. In one particular example, said promotoris the TTRm minimal promoter as defined by SEQ ID NO.6. In analternative embodiment, said liver-specific promoter is the AATpromoter, such as the promoter defined by SEQ ID NO.64. It has beenshown herein that said specific combinations of liver-specificregulatory elements resulted in unexpectedly enhanced liver-specificexpression of the transgene, in particular the FIX transgene or FVIIItransgene described herein, operably linked thereto

As used herein, the terms “identity” and “identical” and the like referto the sequence similarity between two polymeric molecules, e.g.,between two nucleic acid molecules, e.g., two DNA molecules. Sequencealignments and determination of sequence identity can be done, e.g.,using the Basic Local Alignment Search Tool (BLAST) originally describedby Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2sequences” algorithm described by

Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250). Typically,the percentage sequence identity is calculated over the entire length ofthe sequence. As used herein, the term “substantially identical” denotesat least 90%, preferably at least 95%, such as 95%, 96%, 97%, 98% or99%, sequence identity.

The term “functional fragment” as used in the application refers tofragments of the sequences disclosed herein that retain the capabilityof regulating liver-specific expression, i.e. they still confer tissuespecificity and they are capable of regulating expression of a(trans)gene in the same way (although possibly not to the same extent)as the sequence from which they are derived. Fragments comprise at least10 contiguous nucleotides from the sequence from which they are derived.In further particular embodiments, fragments comprise at least 15, atleast 20, at least 25, at least 30, at least 35 or at least 40contiguous nucleotides from the sequence from which they are derived.Also preferably, functional fragments may comprise at least 1, morepreferably at least 2, at least 3, or at least 4, even more preferablyat least 5, at least 10, or at least 15, of the transcription factorbinding sites (TFBS) that are present in the sequence from which theyare derived.

As used in the application, the term “promoter” refers to nucleic acidsequences that regulate, either directly or indirectly, thetranscription of corresponding nucleic acid coding sequences to whichthey are operably linked (e.g. a transgene or endogenous gene). Apromoter may function alone to regulate transcription or may act inconcert with one or more other regulatory sequences (e.g. enhancers orsilencers). In the context of the present application, a promoter istypically operably linked to regulatory elements to regulatetranscription of a transgene.

When a regulatory element as described herein is operably linked to botha promoter and a transgene, the regulatory element can (1) confer asignificant degree of liver specific expression in vivo (and/or inhepatocytes/hepatic cell lines in vitro) of the transgene, and/or (2)can increase the level of expression of the transgene in the liver(and/or in hepatocytes/hepatocyte cell lines in vitro).

According to a particular embodiment, the promoter contained in thenucleic acid expression cassettes and vectors disclosed herein is aliver-specific promoter. This is to increase liver specificity and/oravoid leakage of expression in other tissues. According to a furtherparticular embodiment, the liver-specific promoter is from thetransthyretin (TTR) gene or from the Alpha-1-antitrypsin (AAT) gene.According to yet a further particular embodiment, the TTR promoter is aminimal promoter (also referred to as TTRm or TRRmin herein), mostparticularly the minimal TTR promoter as defined in SEQ ID NO: 6.According to yet a further particular embodiment, the AAT promoter is asdefined in SEQ ID NO: 64.

According to particular embodiments, the promoter in the nucleic acidexpression cassettes and vectors disclosed herein is a minimal promoter.

A ‘minimal promoter’ as used herein is part of a full-size promoterstill capable of driving expression, but lacking at least part of thesequence that contributes to regulating (e.g. tissue-specific)expression. This definition covers both promoters from which(tissue-specific) regulatory elements have been deleted- that arecapable of driving expression of a gene but have lost their ability toexpress that gene in a tissue-specific fashion and promoters from which(tissue-specific) regulatory elements have been deleted that are capableof driving (possibly decreased) expression of a gene but have notnecessarily lost their ability to express that gene in a tissue-specificfashion. Minimal promoters have been extensively documented in the art,a non-limiting list of minimal promoters is provided in thespecification.

The term “liver-specific promoter” encompasses any promoter that confersliver-specific expression to a (trans)gene. Non-limiting examples ofliver-specific promoters are provided on the Liver Specific GenePromoter Database (LSPD, http://rulai.cshl.edu/LSPD/), and include, forexample, the transthyretin (TTR) promoter or TTR-minimal promoter(TTRm), the alpha 1-antitrypsin (AAT) promoter, the albumin (ALB)promotor or minimal promoter, the apolipoprotein A1 (APOA1) promoter orminimal promoter, the complement factor B (CFB) promoter, theketohexokinase (KHK) promoter, the hemopexin (HPX) promoter or minimalpromoter, the nicotinamide N-methyltransferase (NNMT) promoter orminimal promoter, the (liver) carboxylesterase 1 (CES1) promoter orminimal promoter, the protein C (PROC) promoter or minimal promoter, theapolipoprotein C3 (APOC3) promoter or minimal promoter, themannan-binding lectin serine protease 2 (MASP2) promoter or minimalpromoter, the hepcidin antimicrobial peptide (HAMP) promoter or minimalpromoter, and the serpin peptidase inhibitor, clade C (antithrombin),member 1 (SERPINC1) promoter or minimal promoter.

In particularly preferred embodiments, the promoter is a mammalianliver-specific promoter, in particular a murine or human liver-specificpromoter. More preferably, said promoters are the respective minimalpromoters

The term “liver-specific expression” as used in the application, refersto the preferential or predominant expression of a (trans)gene (as RNAand/or polypeptide) in the liver, in liver tissue or in liver cells, ascompared to other (i.e. non-liver) tissues or cells. According toparticular embodiments, at least 50%, more particularly at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99% or 100% of the (trans)geneexpression occurs within liver tissue or liver cells. According to aparticular embodiment, liver-specific expression entails that there isno ‘leakage’ of expressed gene product to other organs or tissue thanliver, such as lung, muscle, brain, kidney and/or spleen. The sameapplies mutatis mutandis for hepatocyte-specific expression andhepatoblast-specific expression, which may be considered as particularforms of liver-specific expression. Throughout the application, whereliver-specific is mentioned in the context of expression,hepatocyte-specific expression and hepatoblast-specific expression arealso explicitly envisaged.

As used herein, the term “liver cells” encompasses the cellspredominantly populating the liver and encompasses mainly hepatocytes,oval cells, liver sinusoidal endothelial cells (LSEC) and cholangiocytes(epithelial cells forming the bile ducts).

The term “hepatocyte,” as used herein, refers to a cell that has beendifferentiated from a progenitor hepatoblast such that it is capable ofexpressing liver-specific phenotype under appropriate conditions. Theterm “hepatocyte” also refers to hepatocytes that are de-differentiated.The term includes cells in vivo and cells cultured ex vivo regardless ofwhether such cells are primary or passaged.

The term “hepatoblast” as used herein, refers to an embryonic cell inthe mesoderm that differentiates to give rise to a hepatocyte, an ovalcell, or a cholangiocyte. The term includes cells in vivo and cellscultured ex vivo regardless of whether such cells are primary orpassaged.

The term “transgene” or “(trans)gene” as used herein refers toparticular nucleic acid sequences encoding a polypeptide or a portion ofa polypeptide to be expressed in a cell into which the nucleic acidsequence is inserted. However, it is also possible that transgenes areexpressed as RNA, typically to lower the amount of a particularpolypeptide in a cell into which the nucleic acid sequence is inserted.These RNA molecules include but are not limited to molecules that exerttheir function through RNA interference (shRNA, RNAi), micro-RNAregulation (miRNA), catalytic RNA, antisense RNA, RNA aptamers, etc. Howthe nucleic acid sequence is introduced into a cell is not essential tothe invention, it may for instance be through integration in the genomeor as an episomal plasmid. Of note, expression of the transgene may berestricted to a subset of the cells into which the nucleic acid sequenceis inserted. The term ‘transgene’ is meant to include (1) a nucleic acidsequence that is not naturally found in the cell (i.e., a heterologousnucleic acid sequence); (2) a nucleic acid sequence that is a mutantform of a nucleic acid sequence naturally found in the cell into whichit has been introduced ; (3) a nucleic acid sequence that serves to addadditional copies of the same (i.e., homologous) or a similar nucleicacid sequence naturally occurring in the cell into which it has beenintroduced ; or (4) a silent naturally occurring or homologous nucleicacid sequence whose expression is induced in the cell into which it hasbeen introduced. By ‘mutant form’ is meant a nucleic acid sequence thatcontains one or more nucleotides that are different from the wild-typeor naturally occurring sequence, i.e., the mutant nucleic acid sequencecontains one or more nucleotide substitutions, deletions, and/orinsertions. In some cases, the transgene may also include a sequenceencoding a leader peptide or signal sequence such that the transgeneproduct will be secreted from the cell.

Typically, the transgenes in the expression cassettes and vectorsdescribed herein encode coagulation factor IX or coagulation factorVIII.

The term “coagulation factor IX” has the meaning as known in the art.Synonyms of coagulation factor IX are “FIX” or “Christmas factor” or“F9” and can be used interchangeably.

In particular, the term “coagulation factor IX” encompasses the humanprotein encoded by the mRNA sequence as defined in Genbank accessionnumber NM_000133.

Preferably, said FIX is a mutated FIX, which is hyperactive orhyper-functional as compared to the wild type FIX. Modifying functionalactivity of human coagulation factor can be done by bioengineering e.g.by introduction of point mutations. By this approach a hyperactive R338Avariant was reported, which showed a 3 fold increased clotting activitycompared to the wild type human FIX in an in vitro activated partialthromboplastin time assay (APPT) (Chang et al., 1998) and a 2 to 6-foldhigher specific activity in hemophilia B mice transduced with the mutantFIX gene (Schuettrumpf et al., 2005). Further exemplary FIXpoint-mutants or domain exchange mutants with even higher clottingactivities have been described: FIX, with the EGF-1 domain replaced withthe EGF-1 domain from FVII, alone or in combination with a R338A pointmutation (Brunetti-Pierri et al., 2009), the V86A/E277A/R338A triplemutant (Lin et al., 2010), the Y259F, K265T, and/or Y345T single, doubleor triple mutants (Milanov, et al., 2012), and the G190V point mutant(Kao et al., 2010), all incorporated herein by reference. In aparticularly preferred embodiment, the FIX mutant is the one describedby Simioni et al., in 2009 and denominated as the “factor IX Padua”mutant, causing X-linked thrombophilia. Said mutant factor IX ishyperactive and carries an R338L amino acid substitution. In a preferredembodiment of the present invention, the FIX transgene used in nucleicacid expression cassettes and expression vectors described hereinencodes the human FIX protein, most preferably the FIX transgene encodesfor the Padua mutant of the human FIX protein.

Accordingly, in a particularly preferred embodiment of the presentinvention, the transgene has SEQ ID NO:9 (i.e. codon-optimized transgeneencoding for the Padua mutant of the human FIX protein).

The term “coagulation factor VIII” has the meaning as known in the art.Synonyms of coagulation factor VIII are “FVIII” or “anti-hemophilicfactor” or “AHF” and can be used interchangeably herein. The term“coagulation factor VIII” encompasses, for example, the human proteinhaving the amino acid sequence as defined in Uniprot accession numberP00451.

In embodiments, said FVIII is a FVIII wherein the B domain is deleted(i.e. B domain deleted FVIII, also referred to as BDD FVIII or FVIIIABor FVIIIdeltaB herein). The term “B domain deleted FVIII” encompassesfor example, but without limitation, FVIII mutants wherein whole or apart of the B domain is deleted and FVIII mutants wherein the B domainis replaced by a linker. Non-limiting examples of B domain deleted FVIIIare described in Ward et al. (2011) and WO 2011/005968, which arespecifically incorporated by reference herein.

In preferred embodiments, said FVIII is B domain deleted FVIII whereinthe B domain is replaced by a linker having the following sequence:SFSQNPPVLTRHQR (SEQ ID NO: 59) (i.e. SQ FVIII as defined in Ward et al.(2011)). In particularly preferred embodiments, said transgene encodingFVIII has SEQ ID NO:18 (i.e. codon-optimized transgene encoding B domaindeleted human FVIII, also referred to herein as (h)FVIIIcopt orco(h)FVIIIdeltaB or co(h)FVIIIdeltaB transgene), as disclosed also in WO2011/005968.

Other sequences may be incorporated in the nucleic acid expressioncassette disclosed herein as well, typically to further increase orstabilize the expression of the transgene product (e.g. introns and/orpolyadenylation sequences).

Any intron can be utilized in the expression cassettes described herein.The term “intron” encompasses any portion of a whole intron that islarge enough to be recognized and spliced by the nuclear splicingapparatus. Typically, short, functional, intron sequences are preferredin order to keep the size of the expression cassette as small aspossible which facilitates the construction and manipulation of theexpression cassette. In some embodiments, the intron is obtained from agene that encodes the protein that is encoded by the coding sequencewithin the expression cassette. The intron can be located 5′ to thecoding sequence, 3′ to the coding sequence, or within the codingsequence. An advantage of locating the intron 5′ to the coding sequenceis to minimize the chance of the intron interfering with the function ofthe polyadenylation signal. In embodiments, the nucleic acid expressioncassette disclosed herein further comprises an intron. Non-limitingexamples of suitable introns are Minute Virus of Mice (MVM) intron,beta-globin intron (betalVS-II), factor IX (FIX) intron A, Simian virus40 (SV40) small-t intron, and beta-actin intron. Preferably, the intronis an MVM intron, more preferably the MVM mini-intron as defined by SEQID NO: 8. The cloning of the MVM intron into a nucleic acid expressioncassette described herein was shown to result in unexpectedly highexpression levels of the transgene operably linked thereto.

Any polyadenylation signal that directs the synthesis of a polyA tail isuseful in the expression cassettes described herein, examples of thoseare well known to one of skill in the art. Exemplary polyadenylationsignals include, but are not limited to, polyA sequences derived fromthe Simian virus 40 (SV40) late gene, the bovine growth hormone (BGH)polyadenylation signal, the minimal rabbit f3-globin (mRBG) gene, andthe synthetic polyA (SPA) site as described in Levitt et al. (1989,Genes Dev 3:1019-1025) (SEQ ID NO:56). Preferably, the polyadenylationsignal is the bovine growth hormone (BGH) polyadenylation signal (SEQ IDNO:10) or the Simian virus 40 (SV40) polyadenylation signal (SEQ IDNO:19).

Typically, the nucleic acid expression cassette according to theinvention comprises a promotor, an enhancer, a (trans)gene, and atranscription terminator.

In a typical embodiment of the present invention, a nucleic acidexpression cassette is disclosed and comprises:

a liver-specific nucleic acid regulatory element, preferably aregulatory element comprising three tandem repeats of the Serpinenhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ IDNO.12,

a liver-specific promoter, and

a transgene.

In a typical embodiment of the present invention, a nucleic acidexpression cassette is disclosed and comprises:

a liver-specific nucleic acid regulatory element, preferably aregulatory element comprising three tandem repeats of the Serpinenhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ IDNO.12,

the liver-specific TTRm promoter (e.g. defined by SEQ ID NO.6, and

a transgene, preferably wherein the combination of the TTRe and TTRmnucleic acids is defined by SEQ ID NO.69.

In a typical embodiment of the present invention, a nucleic acidexpression cassette is disclosed and comprises:

a liver-specific nucleic acid regulatory element, preferably aregulatory element comprising three tandem repeats of the Serpinenhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ IDNO.12,

the liver-specific TTRm promoter, e.g. as defined by SEQ ID NO.6,

an intron, preferably the MVM intron, e.g. as defined by SEQ ID NO.8,and

a transgene preferably wherein the combination of the TTRe and TTRmnucleic acids is defined by SEQ ID NO.69.

In a typical embodiment of the present invention, a nucleic acidexpression cassette is disclosed and comprises:

a liver-specific nucleic acid regulatory element, preferably aregulatory element comprising three tandem repeats of the Serpinenhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ IDNO.12,

the liver-specific TTRm promoter, e.g. as defined by SEQ ID NO.6,

an intron, preferably the MVM intron, e.g. as defined by SEQ ID NO.8,and

a transgene, preferably the FIX or FVIII transgene as defined hereinelsewhere and optionally a transcription terminator as defined hereinelsewhere, preferably wherein the combination of the TTRe and TTRmnucleic acids is defined by SEQ ID NO.69.

In another preferred embodiment of the present invention, a nucleic acidexpression cassette is disclosed and comprises:

a liver-specific nucleic acid regulatory element, preferably aregulatory element comprising three tandem repeats of the Serpinenhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ IDNO.12,

the liver-specific AAT promoter, e.g. as defined by SEQ ID NO.64 , and

a transgene.

In another preferred embodiment of the present invention, a nucleic acidexpression cassette is disclosed and comprises:

a liver-specific nucleic acid regulatory element, preferably aregulatory element comprising three tandem repeats of the Serpinenhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ IDNO.12,

the liver-specific AAT promoter, e.g. as defined by SEQ ID NO.64 , and

an intron, preferably the MVM intron, e.g. as defined by SEQ ID NO.8,and

a transgene.

In another preferred embodiment of the present invention, a nucleic acidexpression cassette is disclosed and comprises:

a liver-specific nucleic acid regulatory element, preferably aregulatory element comprising three tandem repeats of the Serpinenhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ IDNO.12,

the liver-specific AAT promoter, e.g. as defined by SEQ ID NO.64, and

an intron, preferably the MVM intron, e.g. as defined by SEQ ID NO.8,and

a transgene, preferably the FIX or FVIII transgene as defined hereinelsewhere and optionally a transcription terminator as defined hereinelsewhere.

In a typical embodiment of the invention, said nucleic acid expressioncassette disclosed herein comprises:

a liver-specific regulatory element, preferably three tandem repeats ofthe Serpin enhancer (SEQ ID NO.5), e.g. a regulatory element as definedby SEQ ID NO:11,

a promoter, preferably the minimal TTR promoter,

an intron, preferably the MVM intron

a transgene, preferably codon-optimized factor IX cDNA, even morepreferably codon-optimized factor IX Padua cDNA

a transcription terminator, preferably a polyadenylation signal such asthe bovine growth hormone polyadenylation signal (BGHpA) e.g. as definedby SEQ ID NO.10, or the synthetic polyA site as defined by SEQ ID NO:56.

In a further typical embodiment of the present invention, said nucleicacid expression cassette disclosed herein comprises:

a liver-specific regulatory element, preferably three tandem repeats ofthe Serpin enhancer and the transthyretin enhancer (TTRe) (e.g. aregulatory element comprising SEQ ID NO:13, preferably comprising SEQ IDNO:57),

a promoter, preferably the minimal TTR promoter,

an intron, preferably the MVM intron,

a (trans)gene, preferably codon-optimized factor IX cDNA, even morepreferably codon-optimized factor IX Padua cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe bovine growth hormone polyadenylation signal (BGHpA) or thesynthetic polyA site as defined by SEQ ID NO:56.

In another typical embodiment of the present invention, said nucleicacid expression cassette disclosed herein comprises:

a liver-specific regulatory element, preferably a regulatory elementcomprising three tandem repeats of the Serpin enhancer (e.g. aregulatory element comprising SEQ ID NO:11), more preferably threetandem repeats of the Serpin enhancer and the transthyretin enhancer(TTRe) (e.g. a regulatory element comprising SEQ ID NO:13, preferablycomprising SEQ ID NO:57),

a promoter, preferably the minimal TTR promoter,

an intron, preferably the MVM intron,

a (trans)gene, preferably codon-optimized factor VIII cDNA, even morepreferably codon-optimized B domain deleted factor VIII cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe Simian vacuolating virus 40 or Simian virus 40 (SV40)polyadenylation signal or the synthetic polyA site as defined by SEQ IDNO:56.

In another typical embodiment of the present invention, said nucleicacid expression cassette disclosed herein comprises:

a liver-specific regulatory element, preferably a regulatory elementcomprising three tandem repeats of the Serpin enhancer (e.g. SEQ IDNO.5), such as a regulatory element comprising SEQ ID NO:11 and thetransthyretin enhancer (TTRe) as defined by SEQ ID NO.12, such as theregulatory element comprising SEQ ID NO:13, preferably comprising SEQ IDNO:57,

a promoter, preferably the AAT promoter such as the promoter as definedby SEQ ID NO. 64,

an intron, preferably the MVM intron such as the MVM intron as definedby SEQ ID NO.8, a (trans)gene, preferably codon-optimized factor IXcDNA, even more preferably codon-optimized factor IX Padua cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe bovine growth hormone polyadenylation signal (BGHpA) as defined bySEQ ID NO.10, or the synthetic polyA site as defined by SEQ ID NO:56. Asa non-limiting example, such a vector is defined by SEQ ID NO. 65 (cf.FIG. 25).

In another typical embodiment of the present invention, said nucleicacid expression cassette disclosed herein comprises:

a liver-specific regulatory element, preferably a regulatory elementcomprising three tandem repeats of the Serpin enhancer (e.g. SEQ IDNO.5), such as a regulatory element comprising SEQ ID NO:11,

a promoter, preferably the AAT promoter such as the promoter as definedby SEQ ID NO. 64,

an intron, preferably the MVM intron such as the MVM intron as definedby SEQ ID NO.8 , a (trans)gene, preferably codon-optimized factor IXcDNA, even more preferably codon-optimized factor IX Padua cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe bovine growth hormone polyadenylation signal (BGHpA) as defined bySEQ ID NO.10, or the synthetic polyA site as defined by SEQ ID NO:56. Asa non-limiting example, such a vector is defined by SEQ ID NO. 66 (cf.FIG. 25).

In another typical embodiment of the present invention, said nucleicacid expression cassette disclosed herein comprises:

a liver-specific regulatory element, preferably a regulatory elementcomprising the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12,operably linked to three tandem repeats of the Serpin enhancer (e.g. SEQID NO.5), more preferably as defined by SEQ ID NO:11,

a promoter, preferably the AAT promoter such as the promoter as definedby SEQ ID NO. 64,

an intron, preferably the MVM intron such as the MVM intron as definedby SEQ ID NO.8, a (trans)gene, preferably codon-optimized factor IXcDNA, even more preferably codon-optimized factor IX Padua cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe bovine growth hormone polyadenylation signal (BGHpA) as defined bySEQ ID NO.10, or the synthetic polyA site as defined by SEQ ID NO:56. Asa non-limiting example, such a vector is defined by SEQ ID NO.68 (cf.FIG. 25).

The expression cassettes disclosed herein may be used as such, ortypically, they may be part of a nucleic acid vector. Accordingly, afurther aspect relates to the use of a nucleic acid expression cassetteas described herein in a vector, in particular a nucleic acid vector.

In an aspect, the invention also provides a vector comprising a nucleicacid expression cassette as disclosed herein.

The term ‘vector’ as used in the application refers to nucleic acidmolecules, usually double-stranded DNA, which may have inserted into itanother nucleic acid molecule (the insert nucleic acid molecule) suchas, but not limited to, a cDNA molecule. The vector is used to transportthe insert nucleic acid molecule into a suitable host cell. A vector maycontain the necessary elements that permit transcribing the insertnucleic acid molecule, and, optionally, translating the transcript intoa polypeptide. The insert nucleic acid molecule may be derived from thehost cell, or may be derived from a different cell or organism. Once inthe host cell, the vector can replicate independently of, orcoincidental with, the host chromosomal DNA, and several copies of thevector and its inserted nucleic acid molecule may be generated.

The term “vector” may thus also be defined as a gene delivery vehiclethat facilitates gene transfer into a target cell. This definitionincludes both non-viral and viral vectors. Non-viral vectors include butare not limited to cationic lipids, liposomes, nanoparticles, PEG, PEI,etc. Viral vectors are derived from viruses including but not limitedto: retrovirus, lentivirus, adeno-associated virus, adenovirus,herpesvirus, hepatitis virus or the like. Alternatively, gene deliverysystems can be used to combine viral and non-viral components, such asnanoparticles or virosomes (Yamada et al., 2003).

Typically, but not necessarily, viral vectors are replication-deficientas they have lost the ability to propagate in a given cell since viralgenes essential for replication have been eliminated from the viralvector. However, some viral vectors can also be adapted to replicatespecifically in a given cell, such as e.g. a cancer cell, and aretypically used to trigger the (cancer) cell-specific (onco)lysis.Preferred vectors are derived from adeno-associated virus, adenovirus,retroviruses and Antiviruses.

Retroviruses and Antiviruses are RNA viruses that have the ability toinsert their genes into host cell chromosomes after infection.Retroviral and lentiviral vectors have been developed that lack thegenes encoding viral proteins, but retain the ability to infect cellsand insert their genes into the chromosomes of the target cell (Miller,1990; Naldini et al., 1996, VandenDriessche et al., 1999). Thedifference between a lentiviral and a classical Moloney-murineleukemia-virus (MLV) based retroviral vector is that lentiviral vectorscan transduce both dividing and non-dividing cells whereas MLV-basedretroviral vectors can only transduce dividing cells.

Adenoviral vectors are designed to be administered directly to a livingsubject. Unlike retroviral vectors, most of the adenoviral vectorgenomes do not integrate into the chromosome of the host cell. Instead,genes introduced into cells using adenoviral vectors are maintained inthe nucleus as an extrachromosomal element (episome) that persists foran extended period of time. Adenoviral vectors will transduce dividingand nondividing cells in many different tissues in vivo including airwayepithelial cells, endothelial cells, hepatocytes and various tumors(Trapnell, 1993; Chuah et al., 2003). Another viral vector is derivedfrom the herpes simplex virus, a large, double-stranded DNA virus.Recombinant forms of the vaccinia virus, another dsDNA virus, canaccommodate large inserts and are generated by homologous recombination.

Adeno-associated virus (AAV) is a small ssDNA virus which infects humansand some other primate species, not known to cause disease andconsequently causing only a very mild immune response. AAV can infectboth dividing and non-dividing cells and may incorporate its genome intothat of the host cell. These features make AAV a very attractivecandidate for creating viral vectors for gene therapy, although thecloning capacity of the vector is relatively limited. Accordingly, inpreferred embodiments of the invention, the vector used is derived fromadeno-associated virus (i.e. AAV vector).

Different serotypes of AAVs have been isolated and characterized, suchas, for example AAV serotype 2, AAV serotype 5, AAV serotype 8, and AAVserotype 9, and all AAV serotypes are contemplated herein. Inparticular, AAV vectors that comprise a FIX transgene as disclosedherein are preferably AAV serotype 9 vectors, and AAV vectors thatcomprise a FVIII transgene as disclosed herein are preferably AAVserotype 8 vectors.

The AAV vectors disclosed herein may be single-stranded (i.e. ssAAVvectors) or self-complementary (i.e. scAAV vectors). In particular, AAVvectors that comprise a FIX transgene as disclosed herein are preferablyself-complementary, and AAV vectors that comprise a FVIII transgene asdisclosed herein are preferably single-stranded. With the term“self-complementary AAV” is meant herein a recombinant AAV-derivedvector wherein the coding region has been designed to form anintra-molecular double-stranded DNA template.

Gene therapy with adeno-associated viral vectors disclosed herein wasshown to induce immune tolerance towards the transgene comprised in thevector.

In embodiments, the vector according to the invention comprises thefollowing elements (cfr. FIG. 6):

an Inverted Terminal Repeat sequence (ITR), optionally mutated,

a liver-specific regulatory element, preferably a regulatory elementcomprising three tandem repeats of the Serpin enhancer (“Serp” or“SerpEnh”) (e.g. a regulatory element comprising the nucleic acidfragment defined by SEQ ID NO:11),

a promoter, preferably the minimal TTR promoter (TTRm),

an intron, preferably the MVM intron,

a (trans)gene, preferably codon-optimized factor IX cDNA, even morepreferably codon-optimized factor IX Padua cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe BGHpA or the synthetic polyA site as defined by SEQ ID NO:56,

an Inverted Terminal Repeat sequence (ITR).

Preferably, the vector is an adeno-associated virus-derived vector, morepreferably a self-complementary AAV vector, even more preferably aself-complementary AAV serotype 9 vector, such as the vector as definedby SEQ ID NO:2.

In a further typical embodiment of the present invention, said vectorcomprises the following elements (cf. FIG. 8 and FIG. 16):

an Inverted Terminal Repeat sequence (ITR), optionally mutated,

a liver-specific regulatory element, preferably a regulatory elementcomprising three tandem repeats of the Serpin enhancer (“Serp” or“SerpEnh”) and the transthyretin enhancer (TTRe) (e.g. a regulatoryelement comprising SEQ ID NO:13, preferably comprising SEQ ID NO:57),

a promoter, preferably the minimal TTR promoter (TTRm),

an intron, preferably the MVM intron,

a (trans)gene, preferably codon-optimized factor IX cDNA, even morepreferably codon-optimized factor IX Padua cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe BGHpA or the synthetic polyA site as defined by SEQ ID NO:56, and

an Inverted Terminal Repeat sequence (ITR).

The combination of said elements resulted in an unexpectedly highexpression level of FIX, and in particular of the Padua mutant thereof,in the liver of subjects. Preferably, the vector is an adeno-associatedvirus-derived vector, more preferably a self-complementary AAV vector,even more preferably a self-complementary AAV serotype 9 vector, such asthe vector as defined by SEQ ID NO:4 or SEQ ID NO:25.

In another typical embodiment of the present invention, said vectorcomprises the following elements (cfr. FIG. 11, 17 or 18):

an Inverted Terminal Repeat sequence (ITR), optionally mutated,

a liver-specific regulatory element, preferably a regulatory elementcomprising three tandem repeats of the Serpin enhancer (e.g. aregulatory element comprising SEQ ID NO:11),

a promoter, preferably the minimal TTR promoter,

an intron, preferably the MVM intron,

a (trans)gene, preferably codon-optimized factor VIII cDNA, even morepreferably codon-optimized B domain deleted factor VIII cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe Simian vacuolating virus 40 or Simian virus 40 (SV40)polyadenylation signal or the synthetic polyA site as defined by SEQ IDNO:56, and

an Inverted Terminal Repeat sequence (ITR).

The combination of said elements resulted in an unexpectedly highexpression level of FVIII specifically in the liver of subjects.Preferably, the vector is an adeno-associated virus(AAV)-derived vector,more preferably a single-stranded AAV vector, even more preferably asingle-stranded AAV serotype 8 vector, such as the vector as defined bySEQ ID NO:16, SEQ ID NO:20 or SEQ ID NO:21.

In a further embodiment, said vector comprises the following elements(cfr. FIG. 19 or 20):

an Inverted Terminal Repeat sequence (ITR), optionally mutated,

a liver-specific regulatory element, preferably preferably a regulatoryelement comprising three tandem repeats of the Serpin enhancer (“Serp”or “SerpEnh”) and the transthyretin enhancer (TTRe) (e.g. a regulatoryelement comprising SEQ ID NO:13, preferably comprising SEQ ID NO:57),

a promoter, preferably the minimal TTR promoter,

an intron, preferably the MVM intron,

a (trans)gene, preferably codon-optimized factor VIII cDNA, even morepreferably codon-optimized B domain deleted factor VIII cDNA,

a transcription terminator, preferably a polyadenylation signal such asthe Simian vacuolating virus 40 or Simian virus 40 (SV40)polyadenylation signal or the synthetic polyA site as defined by SEQ IDNO:56, and

an Inverted Terminal Repeat sequence (ITR).

The combination of said elements resulted in an unexpectedly highexpression level of FVIII specifically in the liver of subjects.Preferably, the vector is an adeno-associated virus(AAV)-derived vector,more preferably a single-stranded AAV vector, even more preferably asingle-stranded AAV serotype 8 vector, such as the vector as defined bySEQ ID NO:22 or SEQ ID NO:23.

The combination of the triple repeat of the Serpin enhancer defined bySEQ ID NO. 5 and the transthyretin enhancer defined by SEQ ID NO:12including a specific spacer fragment has been shown to be unexpectedlypotent in increasing expression of a transgene operably linked to it.Said regulatory element is defined by SEQ ID NO:13. Said regulatoryelement can further be combined with the transthyretin minimal promotoras defined by SEQ ID NO.6. This creates a combination of 3× the SerpEnh(3× SEQ ID NO.5, e.g. such as in SEQ ID NO.11) with the TTRe and TTRmnucleic acid sequence e.g. as defined by SEQ ID NO.69. For example, sucha construct results in a regulatory element as defined by SEQ ID NO:58,which has been tested to increase the expression of both FVIII and FIXtransgenes as shown herein.

In specific embodiments the following plamids/vectors are provided:pAAVsc-3×CRM8-TTRe-TTRm-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-TTRm-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-TTRm-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-TTRm-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-TTRm-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-TTRm-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-AAT-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-AAT-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-AAT-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-AAT-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-AAT-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-AAT-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-ALBp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-ALBp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-ALBp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-ALBp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-ALBp-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-ALBp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-APOA1p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-APOA1p-MVM-FVIIIcodeltaB-sv40pApAAVsc-TTRe-3×CRM8-APOA1p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-APOA1p-MVM-FVIIIcodeltaB-sv40pApAAVsc-CRM8-TTRe-APOA1p-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-APOA1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-CFBp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-CFBp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-CFBp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-CFBp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-CFBp-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-CFBp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-KHKp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-KHKp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-KHKp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-KHKp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-KHKp-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-KHKp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-HPXp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-HPXp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-HPXp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-HPXp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-HPXp-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-HPXp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-NNMTp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-NNMTp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-NNMTp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-NNMTp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-NNMTp-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-NNMTp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-CES1p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-CES1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-CES1p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-CES1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-CES1p-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-CES1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-PROCp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-PROCp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-PROCp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-PROCp-MVM-FVI I IcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-PROCp-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-PROCp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-APOC3p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-APOC3p-MVM-FVII IcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-APOC3p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-APOC3p-MVM-FVII IcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-APOC3p-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-APOC3p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-MASP2p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-MASP2p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-MASP2p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-MASP2p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-MASP2p-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-MASP2p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-SERPINC1p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-SERPINC1p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-SERPINC1p-MVM-FIXcoR338L-BGHpA,pAAVss-CRM8-TTRe-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-3×CRM8-TTRe-HAMPp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-HAMPp-MVM-FVIIIcodeltaB-sv40pApAAVsc-TTRe-3×CRM8-HAMPp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-HAMPp-MVM-FVIIIcodeltaB-sv40pApAAVsc-CRM8-TTRe-HAMPp-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-HAMPp-MVM-FVIIIcodeltaB-sv40pA.

In any one of said plasmids the poly adenylation signal can be replacedby any other poly adenylation signal such as e.g. the synthetic polyAsite as defined by SEQ ID NO:56 (Synth.pA).

In other embodiments, the vector is a non-viral vector, such as atransposon-based vector. Preferably, said transposon-based vectors arederived from Sleeping Beauty (SB) or PiggyBac (PB). A preferred SBtransposon has been described in Ivics et al. (1997) and its hyperactiveversions, including SB100X, as described in Mates et al. (2009).PiggyBac-based transposons are safe vectors in that they do no enhancethe tumorigenic risk. Furthermore, liver-directed gene therapy withthese vectors was shown to induce immune tolerance towards thetransgene, in particular the hFIX or hFVIII transgene, comprised in thevector.

The transposon-based vectors are preferably administered in combinationwith a vector encoding a transposase for gene therapy. For example, thePiggyBac-derived transposon-based vector can be administered withwild-type PiggyBac transposase (Pbase) or mouse codon-optimized PiggyBactransposase (mPBase) Preferably, said transposases are hyperactivetransposases, such as, for example, hyperactive PB (hyPB) transposasecontaining seven amino acid substitutions (I30V, S103P, G165S, M282V,S509G, N538K, N570S) as described in Yusa et al. (2011), which isspecifically incorporated by reference herein.

Transposon/transposase constructs can be delivered by hydrodynamicinjection or using non-viral nanoparticles to transfect hepatocytes.

In a further aspect, the nucleic acid regulatory elements, the nucleicacid expression cassettes and the vectors described herein can be usedin gene therapy. Gene therapy protocols, intended to achieve therapeuticgene product expression in target cells, in vitro, but also particularlyin vivo, have been extensively described in the art. These include, butare not limited to, intramuscular injection of plasmid DNA (naked or inliposomes), interstitial injection, instillation in airways, applicationto endothelium, intra-hepatic parenchyme, and intravenous orintra-arterial administration (e.g. intra-hepatic artery, intra-hepaticvein). Various devices have been developed for enhancing theavailability of DNA to the target cell. A simple approach is to contactthe target cell physically with catheters or implantable materialscontaining DNA. Another approach is to utilize needle-free, jetinjection devices which project a column of liquid directly into thetarget tissue under high pressure. These delivery paradigms can also beused to deliver viral vectors. Another approach to targeted genedelivery is the use of molecular conjugates, which consist of protein orsynthetic ligands to which a nucleic acid-or DNA-binding agent has beenattached for the specific targeting of nucleic acids to cells (Cristianoet al., 1993).

According to particular embodiments, the use of the nucleic acidexpression cassettes and vectors as described herein is envisaged forgene therapy of liver cells (i.e. liver-directed gene therapy).According to a further particular embodiment, the use of the regulatoryelements, expression cassettes or vectors is for gene therapy, inparticular liver-directed gene therapy, in vivo. According to yet afurther particular embodiment, the use is for a method of gene therapy,in particular liver-directed gene therapy, to treat hemophilia, inparticular to treat hemophilia B or hemophilia A.

Gene transfer into mammalian hepatocytes has been performed using bothex vivo and in vivo procedures. The ex vivo approach requires harvestingof the liver cells, in vitro transduction with long-term expressionvectors, and reintroduction of the transduced hepatocytes into theportal circulation (Kay et al., 1992; Chowdhury et al., 1991). In vivotargeting has been done by injecting DNA or viral vectors into the liverparenchyma, hepatic artery, or portal vein, as well as viatranscriptional targeting (Kuriyama et al., 1991; Kistner et al., 1996).Recent methods also include intraportal delivery of naked DNA (Budker etal., 1996) and hydrodynamic tail vein transfection (Liu et al., 1999;Zhang et al., 1999). According to a further aspect, methods forexpressing a protein in liver cells are provided, comprising the stepsof introducing in liver cells the nucleic acid expression cassette or avector as described herein and expressing the transgene protein productin the liver cells. These methods may be performed both in vitro and invivo.

Methods of gene therapy for a subject in need thereof are also provided,comprising the steps of introducing in the liver of the subject anucleic acid expression cassette containing a transgene encoding atherapeutic protein, and expressing a therapeutic amount of thetherapeutic protein in the liver. According to a further embodiment, themethod comprises the steps of introducing in the liver of the subject avector comprising the nucleic acid expression cassette containing atransgene encoding a therapeutic protein, and expressing a therapeuticamount of the therapeutic protein in the liver.

According to a very specific embodiment, the therapeutic protein encodedby the transgene in the nucleic acid expression cassette or the vectoris factor IX, and the method is a method for treating hemophilia B. Byexpressing factor IX in the liver via gene therapy, hemophilia B can betreated (Snyder et al., 1999). According to another very specificembodiment, the therapeutic protein encoded by the transgene in thenucleic acid expression cassette or the vector is factor VIII, and themethod is a method for treating hemophilia A.

Except when noted differently, the terms “subject” or “patient” are usedinterchangeably and refer to animals, preferably vertebrates, morepreferably mammals, and specifically includes human patients andnon-human mammals, such as e.g. mice. Preferred patients or subjects arehuman subjects.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofproliferative disease, e.g., cancer. Beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilised (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment.

As used herein, a phrase such as “a subject in need of treatment”includes subjects, such as mammalian subjects, that would benefit fromtreatment of a given condition, such as, hemophilia B or hemophilia A.Such subjects will typically include, without limitation, those thathave been diagnosed with the condition, those prone to have or developthe said condition and/or those in whom the condition is to beprevented.

The term “therapeutically effective amount” refers to an amount of acompound or pharmaceutical composition effective to treat a disease ordisorder in a subject, i.e., to obtain a desired local or systemiceffect and performance. In a particular embodiment, the term impliesthat levels of factor IX in plasma equal to or higher than thetherapeutic threshold concentration of 10 mU/ml (milli-units permilliliter) plasma, 50 mU/ml plasma, 100 mU/ml plasma, 150 mU/ml or 200mU/ml plasma in a subject can be obtained by transduction ortransfection of the vector according to any one the embodimentsdescribed herein into a subject. Due to the very high efficiency of thevectors and nucleic acid expression cassettes of the present invention,this high physiological level of factor IX in the subject can beobtained even by administering relatively low doses of vector. Inanother particular embodiment, the term implies that levels of factorVIII in plasma equal to or higher than the therapeutic thresholdconcentration of 10 mU/ml (milli-units per milliliter) plasma, 50 mU/mlplasma, 100 mU/ml plasma, 150 mU/ml plasma, 200 mU/ml plasma or highercan be obtained by transduction or transfection of any of the vectorsdisclosed herein into a subject. Due to the very high efficiency of thevectors and nucleic acid expression cassettes disclosed herein, thesehigh physiological levels of factor VIII in the subject can be obtainedeven by administering relatively low doses of vector. The term thusrefers to the quantity of compound or pharmaceutical composition thatelicits the biological or medicinal response in a tissue, system,animal, or human that is being sought by a researcher, veterinarian,medical doctor or other clinician, which includes alleviation of thesymptoms of the hemophilia being treated. In particular, these termsrefer to the quantity of compound or pharmaceutical compositionaccording to the invention which is necessary to prevent, cure,ameliorate, or at least minimize the clinical impairment, symptoms, orcomplications associated with hemophilia, in particular hemophilia B orhemophilia A, in either a single or multiple dose.

In particular, the transduction of the vector according to any one ofthe embodiments defined herein into the subject can be done at a doselower than 2×10¹¹ vg/kg (viral genomes per kilogram) to obtain atherapeutic factor IX level of 10 mU/ml plasma or of 50 mU/ml plasma ina subject.

Alternatively, if a level of factor IX of 100 mU/ml plasma needs to bereached in a subject, the transduction of the vector according to anyone of the embodiments defined herein into the subject can be done at adose lower than or equal to 6×10¹¹ vg/kg.

Further, if a level of factor IX equal to 150 mU/ml plasma or higherneeds to be reached, the transduction of the vector according to any oneof the embodiments defined herein into the subject can be done ata doselower than or equal than 2×10¹² vg/kg.

In a preferred embodiment, a level of factor IX of 200 mU/ml plasma orhigher can be reached in a subject, when the transduction of the vectoraccording to any one of the embodiments defined herein into the subjectis done at a dose lower than or equal to 2×10¹² vg/kg.

In particular, the transduction of the vector according to any one ofthe embodiments defined herein into the subject can be done at a doselower than or equal to 2×10¹² vg/kg (viral genomes per kilogram), suchas lower than or equal to 1×10¹² vg/kg, 5×10¹¹ vg/kg, 2.5×10¹¹ vg/kg,1×10¹¹ vg/kg, 5×10¹° vg/kg, 1×10¹° vg/kg, 5×10⁹ vg/kg, or 1×10⁹ vg/kgpreferably at a dose lower than or equal to 2.5×10¹¹ vg/kg, to obtain atherapeutic factor VIII level of 10 mU/ml plasma, 50 mU/ml plasma, 100mU/ml plasma, 150 mU/ml plasma, 200 mU/ml plasma, or higher in asubject.

For hemophilia therapy, efficacy of the treatment can, for example, bemeasured by assessing the hemophilia-caused bleeding in the subject. Invitro tests such as, but not limited to the in vitro activated partialthromboplastin time assay (APPT), test factor IX chromogenic activityassays, blood clotting times, factor IX or human factor VIII-specificELISAs are also available. Any other tests for assessing the efficacy ofthe treatment known in the art can of course be used.

The nucleic acid expression cassette, the vector or the pharmaceuticalcomposition of the invention may be used alone or in combination withany of the know hemophilia therapies, such as the administration ofrecombinant or purified clotting factors. The nucleic acid expressioncassette, the vector or the pharmaceutical composition of the inventioncan thus be administered alone or in combination with one or more activecompounds. The latter can be administered before, after orsimultaneously with the administration of the said agent(s).

A further object of the invention are pharmaceutical preparations whichcomprise a therapeutically effective amount of the nucleic acidexpression cassette or the expression vector as defined herein, and apharmaceutically acceptable carrier, i.e., one or more pharmaceuticallyacceptable carrier substances and/or additives, e.g., buffers, carriers,excipients, stabilisers, etc. The term “pharmaceutically acceptable” asused herein is consistent with the art and means compatible with theother ingredients of a pharmaceutical composition and not deleterious tothe recipient thereof. The term “pharmaceutically acceptable salts” asused herein means an inorganic acid addition salt such as hydrochloride,sulfate, and phosphate, or an organic acid addition salt such asacetate, maleate, fumarate, tartrate, and citrate. Examples ofpharmaceutically acceptable metal salts are alkali metal salts such assodium salt and potassium salt, alkaline earth metal salts such asmagnesium salt and calcium salt, aluminum salt, and zinc salt. Examplesof pharmaceutically acceptable ammonium salts are ammonium salt andtetramethylammonium salt. Examples of pharmaceutically acceptableorganic amine addition salts are salts with morpholine and piperidine.Examples of pharmaceutically acceptable amino acid addition salts aresalts with lysine, glycine, and phenylalanine. The pharmaceuticalcomposition according to the invention can be administered orally, forexample in the form of pills, tablets, lacquered tablets, sugar-coatedtablets, granules, hard and soft gelatin capsules, aqueous, alcoholic oroily solutions, syrups, emulsions or suspensions, or rectally, forexample in the form of suppositories.

Administration can also be carried out parenterally, for examplesubcutaneously, intramuscularly or intravenously in the form ofsolutions for injection or infusion. Other suitable administration formsare, for example, percutaneous or topical administration, for example inthe form of ointments, tinctures, sprays or transdermal therapeuticsystems, or the inhalative administration in the form of nasal sprays oraerosol mixtures, or, for example, microcapsules, implants or rods. Thepharmaceutical composition can be prepared in a manner known per se toone of skill in the art. For this purpose, the nucleic acid expressioncassette or the expression vector as defined herein, one or more solidor liquid pharmaceutically acceptable excipients and, if desired, incombination with other pharmaceutical active compounds, are brought intoa suitable administration form or dosage form which can then be used asa pharmaceutical in human medicine or veterinary medicine.

According to another aspect, a pharmaceutical composition is providedcomprising a nucleic acid expression cassette containing a transgeneencoding a therapeutic protein, and a pharmaceutically acceptablecarrier. According to another embodiment, the pharmaceutical compositioncomprises a vector containing the nucleic acid expression cassettecontaining a transgene encoding a therapeutic protein, and apharmaceutically acceptable carrier. According to further particularembodiments, the transgene encodes factor IX and the pharmaceuticalcomposition is for treating hemophilia B or the transgene encodes factorVIII and the pharmaceutical composition is for treating hemophilia A.

The use of the nucleic acid expression cassette, its regulatory elementsand the vector components as disclosed herein for the manufacture ofthese pharmaceutical compositions for use in treating hemophilia,preferably hemophilia B or hemophilia A, is also envisaged.

It is to be understood that although particular embodiments, specificconstructions and configurations, as well as materials, have beendiscussed herein for methods and applications according to the presentinvention, various changes or modifications in form and detail may bemade without departing from the scope and spirit of this invention.

The following examples are provided to better illustrate particularembodiments, and they should not be considered limiting the application.The application is limited only by the claims.

EXAMPLES

Example 1

Design of the AAV Vectors Used

The design of the self-complementary (sc), double-strandedadeno-associated viral (AAV) vectors is depicted in FIG. 1. The minimaltransthyretin promoter (TTRm) is driving the expression of the humancodon-optimized factor IX (co-FIX-R338L) containing thehyper-activating, thrombophilic FIX mutation (R338L). The minimal TTRpromoter (TTRm) is used in conjunction with either the Serpin enhancer(SERP), a triple repeat of the Serpin enhancer (3×SERP), the native TTRenhancer (TTRe), or a combination of a triple repeat of the Serpinenhancer and the native TTR enhancer (3×SERP-TTRe). The minute virus ofmouse intron (MVM), the bovine growth hormone polyadenylation site(BGHpA) or a synthetic polyadenylation site (Synt.pA), and the invertedterminal repeats (ITR) are indicated; 3×SerpEnh means that this elementwas cloned as a triplet repeat upstream of the TTRm. The vector size(including both ITR's) is indicated.

FIG. 2 shows the design of the single-stranded (ss) adeno-associatedviral (AAV) vectors. The minimal transthyretin promoter (TTRm) isdriving the expression of the human codon-optimized B-domain deletedfactor VIII (cohFVIIIdeltaB). Optionally, the Serpin enhancer (SERP), atriple repeat of the Serpin enhancer (3×SERP), the native TTR enhancer(TTRe), or a combination of a triple repeat of the Serpin enhancer andthe native TTR enhancer (3×SERP-TTRe) are cloned upstream of the TTRm.The minute virus of mouse intron (MVM), the Simian Virus 40 (SV40)polyadenylation site (Sv40pA) or a synthetic polyadenylation site(Synt.pA), and the inverted terminal repeats (ITR) are indicated. Thevector size (including both ITR's) is indicated.

Materials and Methods: Cloning Strategy

FIX constructs: The basic AAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpAplasmid (Nair et al. Blood, 2014) was used to create the other AAV-FIXconstructs. This plasmid contains the Serp enhancer (SerpEnh), aliver-specific transthyretin (TTRm) promoter, a minute virus of mouse(MVM) small intron, a codon optimized human FIX transgene containing aR338L mutation, and a bovine growth hormone poly A (BGHpA). The3×SerpEnh-TTRm-MVM sequence was synthesized by GeneArt (LifeTechnologies, Regensburg, Germany) and cloned into the basic constructusing Ascl and Nhel, thereby replacing SerpEnh-TTRm-MVM, creatingpAAVsc-3×SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA. Into this plasmid, the3×SerpEnh sequence was replaced by the TTR enhancer (TTREnh) or3×SerpEnh-TTREnh (constructed by GeneArt), thereby respectively creatingpAAVsc-TTREnh-TTRm-MVM-co-FIX-R338L-BGHpA andpAAVsc-3×SerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L-BGHpA respectively.

FVIII constructs: The basic AAVss-SerpEnh-TTRm-MVM-FVIIIcopt-Sv40pAplasmid was used to create the other AAV-FVIII constructs. This plasmidcontains the Serp enhancer (SerpEnh), a liver-specific transthyretin(TTRm) promoter, an MVM intron, a codon optimized human FVIII transgene(Di Matteo et al., 2014), and an SV40 poly A. The SerpEnh was removedusing Kpnl, followed by re-ligation of the backbone, thereby creatingpAAVss-TTRm-MVM-FVIIIcopt-Sv40pA. To createpAAVsc-3×SerpEnh-TTRm-MVM-FVIIIcopt-Sv40pA, the 3×SerpEnh was firstcloned into a FVIIIcopt plasmid with a different backbone, after whichthe entire expression cassette (3×SerpEnh-TTRm-MVM-FVIIIcopt-Sv40pA) wascloned into the AAVss backbone using Notl and Xhol. From this finalplasmid, 3×SerpEnh was removed and replaced with the TTREnh (constructedby GeneArt), thereby creating pAAVss-TTREnh-TTRm-MVM-FVIIIcopt-Sv40pA.In order to generate pAAVss-3×SerpEnh-TTREnh-TTRm-MVM-cohFVIIIdeltaB-SV40pA (5493bp),pAAVss-TTREnh-TTRm-MVM-cohFVIIIdeltaB-SV40pA (the vector) was restrictedwith Notl-Acc651. A fragment with 3×Serp flanked by Notl/Acc651 wassynthesized by GenArt and ligated into the restricted vector.

AAV Vector Production, Purification and Titration

AAV vectors were produced by calcium phosphate (Invitrogen Corp,Carlsbad, Calif.) co-transfection of AAV-293 human embryonic kidneycarcinoma cells (Stratagene, Carlsbad, Calif., catalog No 240073; withthe pAAV plasmid of interest, an adenoviral helper plasmid and achimeric packaging constructs that delivers the AAV2 Rep gene togetherwith the AAV8 or AAV9 Cap gene, as described previously (VandenDriesscheet al, 2007, VandenDriessche et al; 2007, J. Thromb. Haemost. JTH5:16-24). For the AAV-FIX vectors, the AAV9 serotype was used and forthe AAV-FVIII vectors the AAV8 serotype. The AAV-293 cells are free ofmicrobial contamination as determined by PCR for detection ofmycoplasma. Briefly, two days post transfection, cells were harvestedand vector particles were purified using isopycnic centrifugationmethods. Harvested cells were lysed by successive freeze/thaw cycles andsonication, treated with benzonase (Novagen, Madison, Wis.) anddeoxycholic acid (Sigma-Aldrich, St. Louis, Mo.) and subsequentlysubjected to 2 successive rounds of cesium chloride (Invitrogen Corp,Carlsbad, Calif.) density gradient ultracentrifugation. Fractionscontaining the AAV vector were collected, concentrated in 1mM MgCl₂ inDulbecco's phosphate buffered saline (PBS) (Gibco, BRL) and stored at−80° C. The vector titers (in viral genomes (vg)/ml) were determined byquantitative real-time PCR using specific primers. For the FIX vectors,primers specific for the bovine growth hormone poly A sequence wereused. The forward and reverse primers used were5′-GCCTTCTAGTTGCCAGCCAT-3′ (SEQ ID NO:60) and 5′-GGCACCTTCCAGGGTCAAG-3′(SEQ ID NO:61), respectively. For the FVIII vectors, primers specificfor the FVIII gene sequence were used. The forward and reverse primersused were 5′-AACGGCTACGTGAACAGAAG -3′ (SEQ ID NO:62) and5′-GATAGGGCTGATTTCCAGGC-3′ (SEQ ID NO:63), respectively. Reactions wereperformed in SybrGreen PCR Master Mix (Applied Biosystems, Foster City,Calif., USA), on an ABI 7500 Real-Time PCR System (Applied Biosystems,Foster City,CA,USA). Known copy numbers (102-107) of the respectivevector plasmids used to generate the corresponding AAV vectors, carryingthe appropriate cDNAs were used to generate the standard curves.

Animal Study and Blood Collection

AAV vector administration is carried out by tail vein injection on adultC57B6 mice at two different doses: 1×10⁹ (low dose) and 5×10⁹ (highdose) vector genomes (vg) per mouse as detailed below. Mice were bled atdifferent time points after gene transfer in order to evaluate theFIX/FVIII gene expression. ThepAAVss-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA andpAAVss-3×SerpEnh-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA plasmidconstructs were administered by hydrodynamic delivery into C57BL/6 miceat a dose of 300 ng per mouse. Animals were anesthetized usingisoflurane and blood samples were taken by retro-orbital bleeding ontrisodium citrate. Plasma was prepared immediately after bloodcollection by centrifugation at 14000 rpm for 3 minutes at 4° C. Plasmawas immediately stored at −80° C. for further analysis.

FIX ELISA

The concentration of hFIX antigens in citrated plasma was measured byenzyme-linked immunosorbent assay (ELISA) using manufacturer's protocol(Diagnostica Stago, France). The hFIX standards (available in the kit)were serially diluted using the dilution buffer and used forcalibration. Here the 100% of the standard corresponds to 5000 ng of FIXprotein. The aliquots of the plasma samples were thawed and diluted inorder to make their reading fall in the linear range of standards.Standards and samples were then added to a 96 well plate pre-coated withprimary anti-human FIX antibodies. After an incubation of 1 hour at roomtemperature, the plate was washed and a solution containing secondaryantibodies coupled to peroxidase were added followed by anotherincubation of 1 hour at room temperature. After incubation, theperoxidase substrate TMB (chromogenic solution) was added which resultedin color development. After exact 5 minutes of incubation 1M H2SO4 wasadded to all wells to stop the reaction. After an incubation of15minutes the absorbance was measured at 450 nm using the microplatereader. Using the obtained standard curve, FIX levels were determined.

FVIII ELISA

The concentration of hFVIII antigens in citrated plasma was measured byenzyme-linked immunosorbent assay (ELISA) using manufacturer's protocol(Diagnostica Stago, France).

The hFVIII standards (available in the kit) were serially diluted usingthe dilution buffer and used for calibration. Here the 100% of thestandard corresponds to 200 ng of FVIII protein. The aliquots of theplasma samples were thawed and diluted in order to make their readingfall in the linear range of standards. Standards and samples were thenadded to a 96 well plate pre-coated with primary anti-human FVIIIantibodies. After an incubation of 2 hour at room temperature, the platewas washed and a solution containing secondary antibodies coupled toperoxidase was added followed by another incubation of 2 hour at roomtemperature. After incubation, the peroxidase substrate TMB (chromogenicsolution) was added which resulted in color development. After exact 5minutes of incubation 1M H2504 was added to all wells to stop thereaction. After an incubation of 15 minutes the absorbance was measuredat 450 nm using the microplate reader. Using the obtained standardcurve, FIX levels were determined.

STATISTICS

Data were analyzed using Microsoft Excel Statistics package. Valuesshown in FIG. 3 and FIG. 4 are the mean +SEM. Significance values wereobtained by comparison using t-test.

Viral Administration

Male adult C57BL/6 mice (18-20 grams) were administrated with AAV9 FIXvectors (see the table below) by tail vain injection at doses of 1×10⁹vg/mouse and 5×10⁹ vg/mouse.

Construct Size Dose Mice AAV9sc-SerpEnh-TTRm-MVM-co-FIX- 2510 bp 1 × 10⁹4 R338L-BGHpolyA 5 × 10⁹ 4 AAV9sc-3xSerpEnh-TTRm-MVM-co-FIX- 2682 bp 1 ×10⁹ 4 R338L-BGHpolyA 5 × 10⁹ 4 AAV9sc-TTREnh-TTRm-MVM-co-FIX- 2540 bp 1× 10⁹ 4 R338L-BGHpolyA 5 × 10⁹ 4 AAV9sc-3xSerpEnh-TTREnh-TTRm-MVM- 2760bp 1 × 10⁹ 4 co-FIX-R338L-BGHpolyA 5 × 10⁹ 4

Male adult CB17SCID mice (18-20 grams) were administrated with AAV8XVIII vectors (see the table below) by tail vain injection at doses of1×10⁹ vg/mouse and 5×10⁹ vg/mouse.

Construct/condition Size Dose Mice AAV8ss-TTRm-MVM-FVIIIcopt-sv40pA 5160bp 1 × 10⁹ 4 5 × 10⁹ 4 AAV8ss-SerpEnh-TTRm-MVM-FVIIIcopt- 5252 bp 1 ×10⁹ 4 sv40pA 5 × 10⁹ 4 AAV8ss-3xSerpEnh-TTRm-MVM-FVIIIcopt- 5410 bp 1 ×10⁹ 4 sv40pA 5 × 10⁹ 4 AAV8ss-TTREnh-TTRm-MVM-FVIIIcopt- 5272 bp 1 × 10⁹4 sv40pA 5 × 10⁹ 4

Plasmid Administration

Male adult C57BL/6 mice (22-24 grams) were administrated with respectiveplasmids (see the table below) by hydrodynamic tail vein injection atdoses of 300 ng.

Construct/condition Size Dose Mice pAAVss-TTREnh-TTRm-MVM-co-hFVIII-5272 p  300 ng 2 deltaB-SV40pA pAAVss-3XSerpEnh-TTREnh-TTRm-MVM- 5493 bp300 ng 2 co-hFVIII-deltaB-SV40pA

Results

Factor IX:

The AAV vector containing 3×SerpEnh-TTREnh-TTRm, with 3 copies of theSerp enhancer combined with the natural TTRe enhancer, and incombination with the TTRm promoter, led to the highest FIX levelscompared to any of the other expression cassettes, over 4 time pointsuntil at least day 39 after AAV vector injection. The FIX expressionshowed a continuous increase over time.

The AAV vector containing the most robust 3×SerpEnh-TTREnh-TTRmregulatory elements showed about 11 to 6 fold (at low and high vectordose, respectively) higher FIX expression when compared to a control AAVvector that expressed FIX from the TTREnh-TTRm enhancer/promoter (i.e.lacking the SerpEnh enhancer).

The AAV vector containing the most robust 3×SerpEnh-TTREnh-TTRmregulatory elements showed about 4 to 3 fold (low and high vector dose,respectively) higher FIX expression when compared to a vector thatexpressed FIX from the SerpEnh-TTRm enhancer/promoter, which containsonly one instead of 3 copies of the Serp enhancer, and no TTRe enhancer.

The AAV vector containing the most robust 3×SerpEnh-TTREnh-TTRm showedabout 3 to 2 fold (low and high vector dose, respectively) higherexpression when compared to a vector that expressed FIX from the3×SerpEnh-TTRm enhancer/promoter which also 3 copies of the Serpenhancer, but is devoid of the TTR enhancer.

Factor VIII

The AAV vector containing 3×SerpEnh-TTRm, with 3 copies of the Serpenhancer, led to the highest FVIII levels compared to any of the otherexpression cassettes, until at least day 40 after AAV vector injection.The FVIII expression showed a continuous increase over time.

The AAV vector containing the 3×SerpEnh-TTRm regulatory elements showedabout 30 to 12 fold (low and high vector dose, respectively) higherFVIII expression when compared to a reference control cassette (TTRm)which consist of only the TTR minimal promoter and that do not containany enhancer.

The AAV vector containing the 3×SerpEnh-TTRm regulatory elements showedabout 10 to 1.5-fold (low and high vector dose, respectively) higherFVIII expression when compared to an AAV vector containing SerpEnh-TTRmwith only one copy of the Serp enhancer.

The AAV vector containing the 3×SerpEnh-TTRm regulatory elements showedabout 5 to 2 fold (low and high vector dose, respectively) higherexpression when compared to an AAV vector containing the TTREnh-TTRmenhancer/promoter. The 3×SerpEnh regulatory element could furtherincrease FVIII expression from the TTRenh-TTRm enhancer/promoter (FIG.15). FVIII protein levels were about 8 to 5 fold higher in mice injectedwith the pAAVss-3×SerpEnh-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pAplasmid as compared to thepAAVss-TTREnh-TTRm-MVM-co-hFV111-deltaB-SV40pA plasmid.

Example 2 Studying the In Vivo Effect of 3×CRM8 and TTRenhancer on FVIIIExpression in Various Constructs by Hydrodynamic Injection (2 ml) intoCB17-SCID Mice

The aim of this example was to study the in vivo effect of 3×CRM8 andTTRenhancer on FVIII expression in various constructs by hydrodynamicinjection (2m1) into CB17-SCID mice.

Constructs: The following vectors were compared for FVIII expressionlevels (cf. FIG. 22):

(SEQ ID NO. 14) pAAVss-TTRm-MVM-coFVIIIdeltaB-Sv40pA; (SEQ ID NO: 16)pAAVss-3xSerpEnh-TTRm-MVM-coFVIIIdeltaB-Sv40pA; (SEQ ID NO: 17)pAAVss-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA; and (SEQ ID NO: 22)pAAVss-3xSerpEnh-TTRe-TTRm-MVM-coFVIIIdeltaB- Sv40pA.

In a first experiment, the following conditions were used:

Mouse model: CB17-SCID, male, adult mice of 18-20 grams

Doses: 150 ng plasmid/mouse

Analysis points: Blood collection at Dayl

No of mice: 3 per condition

Design: See the table below

Construct/condition Dose Mice PBS control — 3 1.pAAVss-TTRe-TTRm-MVM-FVIIIcoΔB-sv40pA 300 ng 3 2.pAAVss-3xCRM8-TTRe-TTRm-MVM-FVIIIcoΔB- 300 ng 3 sv40pA

Results: The expression level of FVIII was analysed and compared. FIG.25A example shows a graph depicting the effect of the 3×Serp enhancer onthe TTRenh-TTRm promoter in regulating the FVIII expression in C57BL6mice injected with 300ng of respective plasmids. The FVIII expressionprofile was tested using the ELISA, in the plasma samples collectedDay 1. The presence of 3×Serp enhancer seems to elevate the FVIIIexpression by 8 fold.

In a second experiment the following conditions were used:

Mouse model: CB17-SCID, male, adult mice of 18-20 grams

Doses: 150 ng plasmid/mouse

Analysis points: Blood collection at Dayl

No of mice: 3 per condition

Design: See the table below

Construct/condition Dose Mice PBS control — 3 1.pAAVss-TTRm-MVM-FVIIIcoΔB-sv40pA 150 ng 3 2.pAAVss-3xCRM8-TTRm-MVM-FVIIIcoΔB -sv40pA 150 ng 3 3.pAAVss-TTRe-TTRm-MVM-FVIIIcoΔB-sv40pA 150 ng 3 4.pAAVss-3xCRM8-TTRe-TTRm-MVM-FVIIIcoΔB- 150 ng 3 sv40pA

Results: FIG. 25B shows the compiled data from comparing the 4constructs (see table below for actual FVIII expression values).Addition of 3×SERP to the TTRm construct enhances FVIII expression 17fold. Addition of 3×SERP to the TTRe-TTRm construct enhances FVIIIexpression 13 fold. Addition of TTRe to the 3×SERP-TTRm construct stillenhances FVIII expression 4 fold.

Vectors FVIII level (ng/ml) TTRm 0.68 3xCRM8-TTRm 11.74 TTRe-TTRm 3.523xCRM8-TTRe-TTRm 46.67

Example 3 Studying the In Vivo Effect of 3×CRM8 and TTRenhancer inCombination with an AAT Promoter on FIX Expression in Various Constructsby Hydrodynamic Injection (2 ml) into C57131/6 Mice

In this experiment, the TTRm minimal promotor is replaced by anotherliver-specific promotor to show the versatility of the regulatorelement.

As a first example, the AAT liver-specific promoter (AAT) is tested.

Constructs: Four different constructs were prepared as depicted in FIG.23 (the sequences are depicted in FIG. 24):

(SEQ ID NO: 65) pAAVss-3xSerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA;(SEQ ID NO: 66) pAAVss-3xSerpEnh-AAT-MVM-co-FIX-R338L-BGHpA;(SEQ ID NO: 67) pAAVss-TTRe-AAT-MVM-co-FIX-R338L-BGHpA; and(SEQ ID NO: 68) pAAVss-TTRe-3xSerpEnh-AAT-MVM-co-FIX-R338L-BGHpA;

All constructs tested make use of the AAT promoter (SEQ ID NO.64).

Mouse model: C57BL/6, male, adult mice of 18-20 grams

Doses: 1 μg and 2 μg plasmid/mouse

Analysis points: Blood collection at D1, D2

No of mice: 3 per condition

Design: See the table below

Construct/condition Dose Mice PBS control — 3 1.pAAVsc-3xCRM8-TTRe-AAT-MVM-FIXcoR338L- 1 μg 3 BGHpA 2 μg 3 2.pAAVsc-3xCRM8-AAT-MVM-FIXcoR338L-BGHpA 1 μg 3 2 μg 3 3.pAAVsc-TTRe-AAT-MVM-FIXcoR338L-BGHpA 1 μg 3 2 μg 3 4.pAAVsc-TTRe-3xCRM8-AAT-MVM-FIXcoR338L- 1 μg 3 BGHpA 2 μg 3

Also the corresponding FVIII constructs will be tested for FVIIIexpression with the AAT promotor, hence encompassing the followingconstructs: pAAVss-CRM8-TTRe-AAT-MVM-FVIIIcodeltaB-sv40pA.pAAVss-3×CRM8-AAT-MVM-FVIIIcodeltaB-sv40pA.pAAVss-3×CRM8-TTRe-AAT-MVM-FVIIIcodeltaB-sv40pA andpAAVss-TTRe-3×CRM8-AAT-MVM-FVIIIcodeltaB-sv40pA.

In analogy, the following examples follow the outline of the examplewith the AAT promoter above, but each time with a differentliver-specific promoter or minimal promoter.

In further examples, the albumin promotor (ALBp) is used to replace theTTRm promotor in said constructs, hence encompassing the followingconstructs: pAAVsc-3×CRM8-TTRe-ALBp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-ALBp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-ALBp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-ALBp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-ALBp-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-ALBp-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the apolipoprotein Al promotor (APOA1p) is used toreplace the TTRm promotor in said constructs, hence encompassing thefollowing construct:

pAAVsc-3×CRM8-TTRe-APOA1p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-APOA1p-MVM-FVIIIcodeltaB-sv40pApAAVsc-TTRe-3×CRM8-APOA1p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-APOA1p-MVM-FVIIIcodeltaB-sv40pApAAVsc-CRM8-TTRe-APOA1p-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-APOA1p-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the complement factor B promoter (CFBp) is used toreplace the TTRm promotor in said constructs, hence encompassing thefollowing construct:

pAAVsc-3×CRM8-TTRe-CFBp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-CFBp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-CFBp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-CFBp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-CFBp-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-CFBp-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the ketohexokinase promoter (KHKp) is used toreplace the TTRm promotor in said constructs, hence encompassing thefollowing construct: pAAVsc-3×CRM8-TTRe-KHKp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-KHKp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-KHKp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-KHKp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-KHKp-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-KHKp-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the hemopexin promoter (HPXp) is used to replacethe TTRm promotor in said constructs, hence encompassing the followingconstruct:

pAAVsc-3×CRM8-TTRe-HPXp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-HPXp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-HPXp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-HPXp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-HPXp-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-HPXp-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the nicotinamide N-methyltransferase promoter(NNMTp) is used to replace the TTRm promotor in said constructs, henceencompassing the following construct:pAAVsc-3×CRM8-TTRe-NNMTp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-NNMTp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-NNMTp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-NNMTp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-NNMTp-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-NNMTp-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the (liver) carboxylesterase 1 promoter (CES1p) isused to replace the TTRm promotor in said constructs, hence encompassingthe following construct: pAAVsc-3×CRM8-TTRe-CES1p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-CES1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-CES1p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-CES1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-CES1p-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-CES1p-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the protein C promoter (PROCp) is used to replacethe TTRm promotor in said constructs, hence encompassing the followingconstruct: pAAVsc-3×CRM8-TTRe-PROCp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-PROCp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-PROCp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-PROCp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-PROCp-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-PROCp-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the apolipoprotein C3 promoter (APOC3p) is used toreplace the TTRm promotor in said constructs, hence encompassing thefollowing construct: pAAVsc-3×CRM8-TTRe-APOC3p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-APOC3p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-APOC3p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-APOC3p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-APOC3p-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-APOC3p-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the mannan-binding lectin serine protease 2(MASP2p) is used to replace the TTRm promotor in said constructs, henceencompassing the following construct:pAAVsc-3×CRM8-TTRe-MASP2p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-MASP2p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-MASP2p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-MASP2p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-MASP2p-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-MASP2p-MVM-FVIIIcodeltaB-sv40pA. In further examples,the serpin peptidase inhibitor, clade C (antithrombin) promoter(SERPINC1p) is used to replace the TTRm promotor in said constructs,hence encompassing the following construct:pAAVsc-3×CRM8-TTRe-SERPINC1p-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-TTRe-3×CRM8-SERPINC1p-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-SERPINC1p-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA.

In further examples, the serpin peptidase inhibitor promoter (HAMPp) isused to replace the TTRm promotor in said constructs, hence encompassingthe following construct: pAAVsc-3×CRM8-TTRe-HAMPp-MVM-FIXcoR338L-BGHpA,pAAVss-3×CRM8-TTRe-HAMPp-MVM-FVIIIcodeltaB-sv40pApAAVsc-TTRe-3×CRM8-HAMPp-MVM-FIXcoR338L-BGHpA,pAAVss-TTRe-3×CRM8-HAMPp-MVM-FVIIIcodeltaB-sv40pA,pAAVsc-CRM8-TTRe-HAMPp-MVM-FIXcoR338L-BGHpA, orpAAVss-CRM8-TTRe-HAMPp-MVM-FVIIIcodeltaB-sv40pA.

Results: The expression level of FIX or FVIII will be analysed andcompared as explained in Example 2.

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1. A nucleic acid expression cassette comprising a triple repeat of aliver-specific nucleic acid regulatory element comprising the nucleicacid fragment defined by SEQ ID NO:5 or a sequence having at least 95%identity to said sequence, wherein the liver-specific nucleic acidregulatory elements are operably linked; and a nucleic acid regulatoryelement comprising the nucleic acid fragment defined by SEQ ID NO: 12 ora sequence having at least 95% identity to said sequence; operablylinked to a promoter and a transgene.
 2. The nucleic acid expressioncassette according to claim 1, wherein the promoter is a liver-specificpromoter selected from the group consisting of the minimal TTR promotor(TTRm), the AAT promoter, the albumin (ALB) promotor or minimalpromoter, the apolipoprotein A1 (APOA1) promoter or minimal promoter,the complement factor B (CFB) promoter, the ketohexokinase (KHK)promoter, the hemopexin (HPX) promoter or minimal promoter, thenicotinamide N-methyltransferase (NNMT) promoter or minimal promoter,the (liver) carboxylesterase 1 (CES1) promoter or minimal promoter, theprotein C (PROC) promoter or minimal promoter, the apolipoprotein C3(APOC3) promoter or minimal promoter, the mannan-binding lectin serineprotease 2 (MASP2) promoter or minimal promoter, the hepcidinantimicrobial peptide (HAMP) promoter or minimal promoter, and theserpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1)promoter or minimal promoter.
 3. The nucleic acid expression cassetteaccording to claim 1, wherein said promoter is the minimal TTR promotor(TTRm) as defined by SEQ ID NO:6.
 4. The nucleic acid expressioncassette according to claim 1, wherein said promoter is the AAT promoteras defined by SEQ ID NO:64.
 5. The nucleic acid expression cassetteaccording to claim 1, wherein the liver-specific regulatory elements ofthe triple repeat consist of the the nucleic acid fragment defined bySEQ ID NO:5.
 6. The nucleic acid expression cassette according to 1,wherein said transgene is codon-optimized.
 7. The nucleic acidexpression cassette according to claim 1, wherein said transgene encodescoagulation factor IX (FIX) or wherein said transgene encodescoagulation factor FIX containing a hyper-activating mutation.
 8. Thenucleic acid expression cassette according to claim 1, wherein saidtransgene encodes coagulation factor VIII (FVIII) or wherein saidtransgene encodes coagulation factor VIII having a deletion of the Bdomain.
 9. The nucleic acid expression cassette according to claim 1,wherein the promoter is the transthyretin (TTR) promoter, therebycomprising the combination of the TTRe and TTRm nucleic acids as definedby SEQ ID NO:69.
 10. The nucleic acid expression cassette according toclaim 1, further comprising a minute virus of mouse (MVM) intron. 11.The nucleic acid expression cassette according to claim 1, furthercomprising a transcriptional termination signal derived from the bovinegrowth hormone polyadenylation signal (BGHpA) or derived from the Simianvirus 40 polyadenylation signal (SV40pA), or the syntheticpolyadenylation signal as defined by SEQ ID NO:56.
 12. A vectorcomprising the nucleic acid expression cassette according to claim 1.13. A method of treating hemophilia A or hemophilia B comprisingtransducing or transfecting the vector according to claim 12 into asubject, wherein the vector comprises the FVIII transgene for use intreating hemophilia A or the vector comprises the FIX transgene for usein treating hemophilia B.
 14. The method according to claim 13, whereinafter transduction or transfection of the vector according to claim 12into a subject, levels of factor IX or FVIII in plasma are equal to orhigher than the therapeutic threshold concentration of 10 mU/ml plasmain the subject are obtained.
 15. The method according to claim 14,wherein the transduction of the viral vector into the subject is done ata dose lower than 2×10¹¹ vg/kg.
 16. The method according to claim 15,wherein the transduction of the viral vector into the subject is done ata dose lower than or equal to 6×10¹¹ vg/kg, and wherein levels of factorIX or FVIII in plasma equal to or higher than the therapeuticconcentration of 100 mU/ml are obtained in said subject; or wherein thetransduction of the viral vector into the subject is done at a doselower than or equal to 6×10¹¹ vg/kg, and wherein levels of factor IX orFVIII in plasma equal to or higher than the therapeutic concentration of50 mU/ml are obtained in said subject; or wherein the transduction ofthe viral vector into the subject is done at a dose lower than or equalto 2×10¹² vg/kg, and wherein levels of factor IX or FVIII in plasmaequal to or higher than the therapeutic concentration of 200 mU/ml areobtained in said subject; or wherein the transduction of the viralvector into the subject is done at a dose lower than or equal to 2×10¹²vg/kg, and wherein levels of factor IX or FVIII in plasma equal to orhigher than the therapeutic concentration of 150 mU/ml are obtained insaid subject.
 17. A pharmaceutical composition comprising a vectoraccording to claim 12 and a pharmaceutically acceptable carrier, andoptionally further comprising an active ingredient for treatinghemophilia A when the transgene in said vector is FVIII, or an activeingredient for treating hemophilia B when the transgene in said vectoris FIX.
 18. The nucleic acid expression cassette according to claim 7,wherein the hyper-activating mutation corresponds to an R338L amino acidsubstitution.
 19. The nucleic acid expression cassette according toclaim 8, wherein the B domain of the FVIII is replaced by a linkerdefined by SEQ ID NO:59.
 20. The vector of claim 12, wherein the vectoris a viral vector.